Ramucirumab plus FOLFIRI as second-line treatment for patients with RAS wild-type metastatic colorectal cancer previously treated with anti-EGFR antibody: JACCRO CC-16.

BACKGROUND: Chemotherapy in combination with anti-epidermal growth factor receptor (EGFR) antibody is considered a first-line treatment regimen for RAS wild-type and left-sided metastatic colorectal cancer (mCRC), whereas second-line treatment regimens have not yet been established. Few studies have prospectively evaluated second-line treatment with anti-vascular endothelial growth factor antibody after first-line anti-EGFR antibody therapy for RAS wild-type mCRC. PATIENTS AND METHODS: This non-randomized phase II trial investigated the clinical outcomes of second-line ramucirumab (RAM) plus fluorouracil, levofolinate, and irinotecan (FOLFIRI) after first-line anti-EGFR antibody in combination with doublet or triplet regimen in patients with RAS wild-type mCRC. The primary endpoint was the 6-month progression-free survival (PFS) rate. The secondary endpoints were PFS, overall survival (OS), objective response rate (ORR), rate of early tumor shrinkage (ETS), and safety. We hypothesized a threshold 6-month PFS rate of 30% and an expected 6-month PFS rate of 45%. Treatment was considered effective if the lower limit of the 90% confidence interval (CI) of the 6-month PFS rate was >0.30. RESULTS: Ninety-two patients were enrolled in the study. The primary tumor was located on the left side in 86 (95.6%) patients. Twenty (22.0%) patients had received triplet plus cetuximab as previous therapy. Six-month PFS rate was 58.2% (90% CI 49.3% to 66.2%) with a median PFS of 7.0 months (95% CI 5.7-7.6 months). Median OS was 23.6 months (95% CI 16.5-26.3 months). The ORR and ETS rate were 10.7% and 16.9%, respectively, in 83 patients with measurable lesions. The 6-month PFS rate was comparable between patients previously treated with doublet and triplet regimens; however, median PFS was longer for the doublet regimen (7.4 versus 6.4 months, P = 0.036). CONCLUSIONS: Our study demonstrated prospectively that RAM plus FOLFIRI is an effective second-line treatment after anti-EGFR antibody-containing first-line therapy in RAS wild-type and left-sided mCRC. Furthermore, the results were similar for patients who were previously treated with triplet regimen.

Origins of Vespa velutina hornets that recently invaded Iki Island, Japan and Jersey Island, UK.

The native distribution of the yellow-legged hornet, Vespa velutina, is throughout East Asia. Around 15 years ago this species was accidentally introduced into South Korea and France, where it became established and then spread into neighbouring countries. Previous mitochondrial DNA studies showed that the South Korean, Japanese, and French populations all originated from Eastern China. Recently, the hornet has invaded Iki Island, Japan and Jersey Island, UK. In this study, we analyze the complete mitochondrial DNA sequence of V. velutina to trace the origin of these two populations. The mitochondrial DNA haplotypes (COI, Cytb, and 16S rRNA) of V. velutina in Iki Island matched the unique haplotype present in China, South Korea, and Japan, while the haplotype from Jersey Island matched that of V. velutina found in France and China. These findings were supported by data from the complete mitochondrial DNA sequence of V. velutina from Iki Island, which was consistent with the sequence from South Korea and Tsushima, whereas V. velutina in Jersey was most similar to the French population.

Impact of Donor Age on Outcome of Intestinal Transplantation in Japan.

BACKGROUND: Donor age for intestinal transplantation (ITx) is somewhat younger than that for other solid organs. Clear criteria for adequate donors have not been established. There is a donor scarcity for ITx in Japan due to the shortage of young donors. METHODS: We reviewed outcomes associated with ITx in Japan based on donor age for cadaveric and living donation. RESULTS: Standardized report forms were sent to all known ITx programs, asking for information on ITxs performed between 1996 and 2016. All programs responded. Patient and graft survival estimates were obtained using the Kaplan-Meier method. Five institutions provided data on 27 grafts in 24 patients. There were 14 cadaveric and 13 living donor transplants. Median donor age for ITxs was 40 (range, 17-60) years. Graft survival at 5 years was 66% for patients >40 years old (n = 18) and 47% for those <40 years old (n = 9), not a statistically significant difference (P = .49). Graft survival at 5 years was 60% in those >50 years old (n = 5) and 57% for those <50 years old (n = 22), again not a significant difference (P = .27). CONCLUSION: There is no difference in survival between for those with donor age <40 vs >40 years. Donor age for ITx can be extended from >40 to up to 50 years, which may help to mitigate the donor shortage. It will be necessary to clarify the donor criteria for ITx through accumulation of further data on ITx.

Three-Year Prospective Follow-up of Potential Pediatric Candidate for Intestinal Transplantation.

Intestinal transplantation (ITx) is a treatment for refractory intestinal failure (IF). However, the indications for and timing of ITx are still controversial because the course of IF is unknown. We performed a prospective multi-institutional cohort study to identify the prognostic factors for referral to an ITx facility. Patients under 18 years of age in Japan who suffered from IF and had received parenteral nutrition for longer than 6 months were enrolled in this study. They were followed up for 3 years. Seventy-two patients were followed. The mean age at the beginning of the study was 7.0 years. Diagnoses were short gut syndrome (n = 25), motility disorder (n = 45), and other (n = 2). The overall 3-year survival rate was 95%. The 3-year survival rate was 86% in patients with intestinal-failure-associated liver disease (IFALD) (n = 6) compared to 97% in those without IFALD (n = 66) (P = .0003). Furthermore, the 3-year survival rates of patients who did and did not meet the criteria for ITx were 82% (n = 11) and 97% (n = 62), respectively (P = .034). Six (44%) of 14 patients whose performance status (PS) was ≥3 at enrollment were dead or still had a PS ≥ 3 at 3 years. This study indicates that IFALD is a poor prognostic factor in pediatric patients with IF. Our indication for ITx, namely the presence of IFALD or loss of more than 2 parenteral nutrition access sites, seems to be applicable.

Immunological Response of Pigs to Human Cells, Including Issues Such as the Production of Natural Antibodies in Newborns.

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mother's milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

Everolimus Rescue Treatment for Chronic Rejection After Pediatric Living Donor Liver Transplantation: 2 Case Reports.

Chronic rejection (CR) remains a challenging complication after liver transplantation. Everolimus, which is a mammalian target of rapamycin inhibitor, has an anti-fibrosis effect. We report here the effect of everolimus on CR. Case 1 was a 7-year-old girl who underwent living donor liver transplantation (LDLT) shortly after developing fulminant hepatitis at 10 months of age. Liver function tests (LFTs) did not improve after transplantation despite treatment with tacrolimus + mycophenolate mofetil (MMF). Antithymoglobulin (ATG) and steroid pulse therapy were also ineffective. The patient was diagnosed with CR, and everolimus was started with a target trough level of about 5 ng/mL. LFTs improved and pathological examination showed no progression of hepatic fibrosis. Case 2 was a 10-year-old girl with Alagille syndrome who underwent LDLT at 1 year of age. She had biopsy-proven acute cellular rejection with prolonged LFT abnormalities beginning 3 years after transplantation. She was treated with steroid pulse therapy, followed by MMF, tacrolimus, and prednisolone. Her condition did not improve, even after subsequent ATG administration. CR was suspected based on liver biopsy in the fourth postoperative year, and everolimus was introduced. The target trough level was around 5 ng/mL, but was reduced to 3 ng/mL due to stomatitis. Four years have passed since the initiation of everolimus, and LFTs are stable with no progression of liver biopsy fibrosis. We describe 2 cases in which everolimus was administered for CR. In both cases, LFTs improved and fibrosis did not progress, suggesting that everolimus is an effective treatment for CR after LDLT.

Pulmonary Arterial Pressure Management Based on Oral Medicine for Pediatric Living Donor Liver Transplant With Portopulmonary Hypertension.

Pediatric living donor liver transplantation (LDLT) in patients with advanced portopulmonary hypertension (PoPH) is associated with poor prognoses. Recently, novel oral medications, including endothelin receptor antagonists (ERAs), phosphodiesterase 5 (PDE5) inhibitors, and oral prostacyclin (PGI2) have been used to treat PoPH. Pediatric patients with PoPH who underwent LDLT from 2006 to 2016 were enrolled. Oral pulmonary hypertension (PH) medication was administered to control pulmonary arterial pressure (PAP). Four patients had PoPH. Their ages ranged from 6 to 16 years, and their original diseases were biliary atresia (n = 2), portal vein obstruction (n = 1), and intrahepatic portal systemic shunt (n = 1). For preoperative management, 2 patients received continuous intravenous PGI2 and 2 oral medications (an ERA alone or an ERA and a PDE5 inhibitor), and 2 received only oral drugs (an ERA and a PDE5 inhibitor). One patient managed only with intravenous PGI2 died. In the remaining 3 cases, intravenous PGI2 or NO was discontinued before the end of the first postoperative week. Postoperative medications were oral PGI2 alone (n = 1), an ERA alone (n = 1), or the combination of an ERA and a PDE5 inhibitor (n = 1). An ERA was the first-line therapy, and a PDE5 inhibitor was added if there was no effect. New oral PH medications were effective and safe for use in pediatric patients following LDLT. In particular, these new oral drugs prevent the need for central catheter access to infuse PGI2.

Regulatory T cells with superior immunosuppressive capacity emigrate from the inflamed colon to draining lymph nodes.

Foxp3+ Regulatory T cells (Tregs) play a critical role in the maintenance of colon homeostasis. Here we utilized photoconvertible KikGR mice to track immune cells from the caecum and ascending (proximal) colon in the steady state and DSS-induced colitis. We found that Tregs from the proximal colon (colonic migratory Tregs) migrated exclusively to the distal part of mesenteric lymph nodes (dMLN) in an S1PR1-dependent process. In the steady state, colonic migratory CD25+ Tregs expressed higher levels of CD103, ICOS, LAG3 and CTLA-4 in comparison with pre-existing LN Tregs. Intestinal inflammation led to accelerated Treg replacement in the colon, bidirectional Treg migration from the colon to dMLN and vice versa, as well as increases in Treg number, proliferation and expression of immunosuppressive molecules. This was especially apparent for CD25 very high Tregs induced in colitis. Furthermore, colonic migratory Tregs from the inflamed colon included more interleukin (IL)-10 producing cells, and demonstrated greater inhibition of T-cell proliferation in comparison with pre-existing LN Tregs. Thus, our results suggest that Tregs with superior immunosuppressive capacity are increased both in the colon and dMLN upon inflammation. These Tregs recirculate between the colon and dMLN, and are likely to contribute to the downregulation of intestinal inflammation.

Edoxaban is effective in reducing the incidence of asymptomatic phlebographic events following closed-wedge high tibial osteotomy.

INTRODUCTION: The purpose of this study was twofold: to investigate whether edoxaban significantly decreases the rate of venous thromboembolism (VTE) following closed-wedge high tibial osteotomy (CWHTO), in terms of phlebographic event, and to determine whether edoxaban is safe or increases the rate of hemorrhagic complications. We hypothesized that edoxaban would decrease the incidence of VTE and would not increase the rate of hemorrhagic complications. MATERIALS AND METHODS: We randomly enrolled 60 patients undergoing CWHTO. The patients were divided into two groups: one group receiving edoxaban (15mg in 5 patients, 30mg in 23 patients) and a non-edoxaban group. All patients underwent computed tomography venography on day 7to diagnose postoperative VTE. Blood samples were obtained on the day before CWHTO and on postoperative days 1, 3, 7 and 14. The incidence of VTE and hemorrhagic events in both groups was compared using unpaired Student t-test or chi-square test. RESULTS: The incidence of VTE was significantly greater in the non-edoxaban group (31.3% versus 7.1%; P=0.02). The incidence of deep vein thrombosis (DVT) was also significantly greater in the non-edoxaban group (28.1% versus 3.6%; P=0.01). A single patient from the edoxaban group experienced major bleeding. On days 3 and 7, D-dimer levels were significantly lower in the edoxaban group (P=0.03 and 0.003, respectively). On days 3, 7 and 14, activated partial thromboplastin time was significantly greater in the edoxaban group (P=0.02, 0.01 and 0.006, respectively). CONCLUSION: Patients undergoing CWHTO are at risk of postoperative VTE. Edoxaban helps prevent asymptomatic phlebographic VTE and DVT following CWHTO; however, the risk of major bleeding must be considered. LEVEL OF EVIDENCE: II.

Fibroadenoma in adolescent females after living donor liver transplantation.

Breast FA is the most common breast tumor diagnosed in young women. Female renal transplant recipients on CsA have an increased risk of developing FA. However, reports of FA after LDLT have not been described. Our objectives were to determine the incidence of FA, analyze risk factors for FA, and evaluate treatment strategies in adolescent females after LDLT. A total of 18 female patients aged 10-19 years who underwent LDLT and survived at least one year after transplantation were enrolled in our study. The incidence of FA was 11.1%. To determine pre- or post-transplant conditions that are associated with FA after transplantation, the patients were divided into two groups according to the presence or absence of FA: FA group (n=2) and non-FA group (n=16). There were no differences in mean age at LDLT, mean age at breast evaluation, and mean duration between transplantation and breast evaluation between the two groups. However, there was a difference in the immunosuppressive regimen between the two groups. The FA group was maintained on CsA, whereas the non-FA group was maintained on tacrolimus. CsA might be implicated in FA development in adolescent females after LDLT.

Risk stratification for congenital diaphragmatic hernia by factors within 24 h after birth.

OBJECTIVE: To establish a simple risk stratification system for patients with congenital diaphragmatic hernia (CDH) based on postnatal information within 24 h after birth. STUDY DESIGN: A multi-institutional retrospective cohort study was conducted including 348 neonates who had isolated CDH born between 2006 and 2010. Based on the two most powerful variables for 90-day survival selected by multivariate analyses, a risk stratification system was established. RESULTS: Multiple logistic regression analysis identified two adverse prognostic factors: an Apgar score at 1 min (Ap1) of 0-4 (odds ratio (OR) 3.3, P=0.004), and a best oxygenation index (OI) ⩾8.0 (OR 11.4, P<0.001). Based on a combinations of these two factors, patients were classified into three risk categories. The 90-day survival rates in categories 1-3 were 100, 88 and 52%, respectively (P<0.001). CONCLUSION: Our simple risk stratification system based on Ap1 and best OI was capable of predicting mortality well.

Liver Failure From Ultra-Short Bowel Syndrome on the Intestinal Transplant Waiting List: A Retrospective Study.

BACKGROUND: Patients with intestinal failure (IF) are candidates for intestinal transplantation (ITx). In Japan, these patients have few opportunities to undergo cadaveric ITx because of low rates of organ donation. The donor criteria and recipient priority for ITx are still unknown. We reviewed our cases of IF to investigate which patients should be prioritized for ITx. METHODS: Patients with IF who were registered as candidates for cadaveric ITx between January 2010 and November 2015 in our institute were included in this retrospective study. Their data were gathered from their charts and analyzed. RESULTS: Five patients were included. Their primary diseases included total colon aganglionosis (n = 1), chronic idiopathic intestinal pseudo-obstruction syndrome (n = 2), superior mesenteric vein embolization (n = 1), and graft loss after ITx (n = 1). Two patients died of liver failure (LF) during the waiting period. The remaining three are now alive and waiting for transplantation. The lengths of the remaining intestine were more than 20 cm in living cases but less than 20 cm in fatal cases. In the fatal cases, they had several episodes of catheter-related blood stream infection, which caused LF and acute renal failure. CONCLUSIONS: We identified two patients with less than 20 cm residual small bowel who died after acute deterioration of liver function. Patients with ultra-short bowel could have a higher risk of LF. Therefore, they should be referred as soon as possible to a specialized hospital where ITx is a choice of treatment for IF.

The relationship between three signs of fetal magnetic resonance imaging and severity of congenital diaphragmatic hernia.

OBJECTIVE: To seek a simple approach for prenatally classifying congenital diaphragmatic hernia (CDH) severity using fetal magnetic resonance imaging (MRI) markers. STUDY DESIGN: A retrospective, multicenter study using questionnaires to investigate fetal MRI findings. We included fetuses prenatally diagnosed with isolated left-sided CDH and delivered after 36 weeks of gestation. We focused on three fetal MRI morphological signs: incomplete pulmonary baseline (IPB), liver up (LU) and retrocardiac stomach (RCS). We also evaluated the fetal MRI score defined as the total number of positive signs; the primary outcome was survival at discharge. RESULTS: In 256 patients (from 56 institutions), IPB, LU and RCS findings correlated with lower survival: odds ratio (95% confidence interval), 0.16 (0.08 to 0.33); 0.24 (0.12 to 0.51); and 0.14 (0.07 to 0.28); respectively. Patients with higher fetal MRI scores had a higher mortality rate. CONCLUSION: IPB, LU and RCS on fetal MRI are related to CDH severity.

The induction of MIG6 under hypoxic conditions is critical for dormancy in primary cultured lung cancer cells with activating EGFR mutations.

The biologic activity of individual cancer cells is highly heterogeneous. Hypoxia, one of the prominent features of a tumor microenvironment, is thought to be causal in generating this cellular heterogeneity. In this study, we revealed that primary lung cancer cells harboring activating epidermal growth factor receptor (EGFR) mutations generally entered a dormant state when hypoxic. We found that heterodimer formation of the ERBB family receptor tyrosine kinases (RTKs), and their subsequent downstream signaling, was diminished under hypoxic conditions, although phosphorylation of the EGFR was retained. Dormant lung cancer cells were found to be resistant to EGFR tyrosine kinase inhibitor (TKI) treatment. In terms of mechanism, we found that a negative regulator of ERBB signaling, MIG6/ERRFI1/RALT/Gene33, was induced by hypoxia both in vitro and in vivo. MIG6 expression prevented heterodimer formation of ERBB family RTKs, and suppressed their downstream signaling. Knockdown of MIG6 enhanced tumor cell growth under hypoxic conditions, and promoted the phosphorylation of ERK and AKT via increased EGFR-HER3 binding. Critically, sensitivity to an EGFR-TKI, as well as to irradiation under hypoxic conditions, was increased in MIG6 knockdown cells. The expression of MIG6 was partly correlated with a pS6 negative zone in patient tumors. Analyses of tumor sections from 68 patients with activating EGFR mutations showed that patients with high MIG6 expression showed significantly shorter survival after EGFR-TKI treatment than other groups. Collectively, our data suggest that dormant cancer cells with a high MIG6 expression level might be one of the causes of EGFR-TKI resistance in EGFR mutant lung cancer cells.

Vibrational spectroscopic evidence for (NO)3 formation on Cu(111).

The formation of (NO)3 on Cu(111) was recently reported based on scanning tunneling microscopy observations [A. Shiotari et al., J. Chem. Phys. 141, 134705 (2014)]. We herein report studies into this system using electron energy loss spectroscopy and verify the above findings through vibrational analysis. For the surface covered with mixed isotopes of N(16)O and N(18)O, we observed four peaks corresponding to N-O stretching vibrations, which were ascribed to the four isotopic combinations of the trimer. Dynamic coupling within the trimer was evaluated from model calculations of the coupled oscillators. Furthermore, we observed hindered rotation and translation modes in the dipole scattering regime, suggesting that the molecular axis is tilted from the surface normal. These results provide spectroscopic support for the formation of (NO)3 on Cu(111).

Adsorbed states of chlorophenol on Cu(110) and controlled switching of single-molecule junctions.

A molecular junction of substituted benzene (chlorophenol) is fabricated and controlled by using a scanning tunneling microscope (STM). Prior to the junction formation, the bonding geometry of the molecule on the surface is characterized by STM and electron energy loss spectroscopy (EELS). EELS shows that the OH group of chlorophenol is dissociated on Cu(110) and that the molecule is bonded nearly flat to the surface via an O atom, with the Cl group intact. We demonstrate controlled contact of an STM tip to the "available" Cl group and lift-up of the molecule while it is anchored to the surface via an O atom. The asymmetric bonding motifs of the molecule to the electrodes allow for reversible control of the junction.

Impact of Donor-Specific Antibodies on Graft Fibrosis After Pediatric Living Donor Liver Transplantation for Biliary Atresia.

BACKGROUND: Pediatric living donor liver transplant (LDLT) patients sometimes develop graft fibrosis after non-recurrent diseases such as biliary atresia (BA). Donor-specific antibodies (DSA) have recently been shown to play a possible role in graft damage after liver transplantation. We report the impact of DSA on pediatric LDLT for BA patients. METHODS: Patients under age 18 years who received LDLT for BA at our institution and who had at least 5 years' follow-up were identified, and 23 were eventually enrolled in this study. Pathological findings were assessed with the use of the last available biopsy. Patients were divided into 2 groups, DSA-positive and DSA-negative. Graft fibrosis after LDLT was assessed according to DSA groups. RESULTS: The mean patient age at transplant was 2.6 years. The mean time to the last available biopsy after LDLT was 8.2 years (4.8-15.6 years); 6 patients (26%) showed no fibrosis, whereas fibrosis was graded as F1, F2, or F3 in 8 patients (35%), 8 patients (35%), and 1 patient, respectively. DSA were observed in 12 patients (52%). Moderate graft fibrosis (F2 and F3) was found in 7 (58%) of the DSA-positive group, but only 2 (18%) of the DSA-negative group, showing a statistically significant difference (P < .05). Pre-transplant cross-matching was performed in 17 patients. The 2 patients with a positive cross-match were DSA-positive. Six cross-match-negative patients developed de novo DSA after LDLT. CONCLUSIONS: Graft fibrosis was observed after LDLT for BA during long-term follow-up, more commonly in DSA-positive patients. DSA may play a role in fibrosis formation.

Expression of a Synthetic Gene of CTDM by Transgenic Animals.

BACKGROUND: The purpose of this study was to produce molecules that can precisely regulate the complement and coagulation system and to assess the expression of such molecules in transgenic animals. METHODS: The CTDM gene, which is composed of the delta-1-99 amino acid (aa) C1-INH, EGF domain 4-6 of thrombomoduline (TM), short consensus repeat (SCR) 2-4 of DAF(CD55), and SCR 2-4 of MCP(CD46) was established. The codon usage for expression in mammals was adopted. The cDNA of CTDM was subcloned into the pCPI site (the human insulin promoter and a cytomegalovirus enhancer). pCPI-CTDM was transfected into pig endothelial cells (PEC). The expression of the molecule was clearly assessed by means of flow cytometry. RESULTS: BD3F1 female mice were induced to superovulate and were then crossed with BD3F1 males. Micro-injection and embryo transfer were performed by standard methods, thus generating transgenic mice that express CTDM. The mice carried the CTDM plasmid, as verified by PCR. Tissue expression levels in transgenic mouse lines generated with the constructs were follows: pancreas, 1.0; brain, 5.4; thymus, 0.3; heart, 0.2; lung, 1.2; liver, 0.1; kidney, 0.1; intestine, 0.4; and spleen, 1.6. A naive control mouse was also analyzed in the exact manner as for the transgenic mice. CONCLUSIONS: A synthetic CTDM gene with codon usage optimized to the mammalian system represents a critical factor in the development of transgenic animals.

Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components.

BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

HLA-G1, but Not HLA-G3, Suppresses Human Monocyte/Macrophage-mediated Swine Endothelial Cell Lysis.

The inhibitory function of HLA-G1, a class Ib molecule, on monocyte/macrophage-mediated cytotoxicity was examined. The expression of inhibitory receptors that interact with HLA-G, immunoglobulin-like transcript 2 (ILT2), ILT4, and KIR2DL4 (CD158d) on in vitro-generated macrophages obtained from peripheral blood mononuclear cells and the phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells were examined by flow cytometry. cDNAs of HLA-G1, HLA-G3, HLA-E, and human ß2-microglobulin were prepared, transfected into pig endothelial cells (PECs), and macrophage- and the THP-1 cell-mediated PEC cytolysis was then assessed. In vitro-generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on PEC indicated a significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. HLA-G1 was clearly expressed on the cell surface of PEC, whereas the levels of HLA-G3 were much lower and remained in the intracellular space. On the other hand, the PMA-activated THP-1 cell was less expressed these inhibitory molecules than in vitro-generated macrophages. Therefore, the HLA-G1 on PECs showed a significant but relatively smaller suppression to THP-1 cell-mediated cytotoxicity compared to in vitro-generated macrophages. These results indicate that by generating HLA-G1, but not HLA-G3, transgenic pigs can protect porcine grafts from monocyte/macrophage-mediated cytotoxicity.