Hao-Chih Tai

In vitro investigation of pig cells for resistance to human antibody-mediated rejection.

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.

Recent Trend of Necrotizing Fasciitis in Taiwan: Focus on Monomicrobial Klebsiella pneumoniae Necrotizing Fasciitis

BACKGROUND Necrotizing fasciitis (NF) is a rapidly progressive, life-threatening soft-tissue infection that is traditionally caused by group A Streptococcus (GAS) or mixed aerobic/anaerobic bacteria. Monomicrobial Klebsiella pneumoniae NF (KP-NF) has been reported since 1996 but has not yet been systematically studied. METHODS We retrospectively studied consecutive NF cases treated at a university hospital in Taiwan during 1997-2010 and investigated the clinical characteristics and outcomes associated with monomicrobial KP-NF, using monomicrobial GAS-NF as a reference. We also analyzed the virulence gene profiles of the isolated K. pneumoniae strains. RESULTS Of 134 NF cases, 88 were monomicrobial, of which the most common pathogens were GAS (n = 16) and K. pneumoniae (n = 15). Monomicrobial KP-NF entailed a moderate risk of limb loss (20% vs 25%; P = 1.000) and high mortality (47% vs 19%; P = .135), and it was more likely to involve bacteremia (80% vs 31%; P = .011), concomitant distant abscesses (27% vs 0%; P = .043), and underlying immunocompromising conditions (100% vs 63%; P = .018), compared with GAS-NF. The isolated K. pneumoniae strains (n = 10) were of capsular polysaccharides genotype K1 (n = 4), K54/K20/K5 (n = 4), K2 (n = 1), and K16 (n = 1). All strains carried rmpA, iucABCDiutA, and iroA. Genotype K1 strains had a significantly higher risk of concomitant distant abscesses, compared with non-K1 strains (75% vs 0%; P = .033). CONCLUSIONS K. pneumoniae has become a common pathogen of monomicrobial NF in Taiwan. Physicians treating patients with monomicrobial KP-NF should be aware of the risk of concomitant distant abscesses, particularly in cases caused by genotype K1.

Progress in xenotransplantation following the introduction of gene-knockout technology.

The production of α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pigs has overcome the barrier of preformed anti‐Galα1,3Gal (Gal) antibodies that has inhibited progress in pig‐to‐primate organ xenotransplantation for many years. Survival of GT‐KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti‐nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild‐type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T‐cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ‐source pigs is therefore required to overcome these problems. GT‐KO pigs expressing a human complement‐regulatory protein, e.g. decay‐accelerating factor, and/or an ‘anti‐coagulant’ gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti‐coagulant to a procoagulant phenotype, and protect the islets from early loss.

Suppressive efficacy and proliferative capacity of human regulatory T cells in allogeneic and xenogeneic responses.

Background. An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro. Methods. Human peripheral blood mononuclear cells (PBMC), CD4+T cells, or CD4+CD25−T cells were stimulated with irradiated human or wild type (WT) or &agr;1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+CD25highTregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xeno-reacitve Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis. Results. The responses of human PBMC, CD4+T cells, and CD4+CD25−T cells to pig PBMC were stronger than to human PBMC (P<0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P<0.05). Human CD4+CD25highTregs suppressed proliferation of CD4+CD25−T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+CD25+FoxP3high responder T cells proliferated more than their xenoreactive counterparts (P<0.05), although xenoreactive CD4+CD25+T cells proliferated more than alloreactive cells (P<0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+CD25+FoxP3high cells. Conclusions. Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+CD25+FoxP3high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.

Late onset of development of natural anti‐nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants

If an ABO‐incompatible heart is transplanted into an infant before natural antibodies have developed to the specific donor carbohydrate A/B antigen(s), then B‐cell tolerance to the donor A/B antigen is achieved, and these antibodies never develop. Anti‐carbohydrate antibodies play a role in the rejection of wild type (WT) and α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pig xenografts. We investigated development of these antibodies in infant baboons and humans. Serum samples from infant baboons (n = 42) and humans (n = 42) were tested by flow cytometry for immunoglobulin M and immunoglobulin G binding to peripheral blood mononuclear cells from WT and GT‐KO pigs, and for complement‐dependent cytotoxicity. The presence of anti‐blood group antibodies was tested in baboon serum. In infant baboons and humans, cytotoxic anti‐Galα1,3Gal antibodies develop during the first 3 months, and steadily increase with age, whereas cytotoxic anti‐nonGal antibodies are either absent or minimal in the majority of cases throughout the first year of life. Anti‐blood group antibodies were not detected before 16 weeks of age. Our data suggest GT‐KO pig organ/cell transplants could be carried out in early infancy in the absence of preformed cytotoxic anti‐nonGalα1,3Gal antibodies.

Safe induction of diabetes by high-dose streptozotocin in pigs.

Objectives: Streptozotocin (STZ) has been widely used to induce diabetes in rodents and nonhuman primates, but it has been found difficult to achieve a completely diabetic state in pigs in the absence of detrimental side effects. As a result, pancreatectomy has been advocated in this species. We have investigated the effects of 2 dosages of STZ to safely induce diabetes in pigs. Methods: Three pigs received Zanosar STZ at 150 mg/kg (group 1). Four pigs received Zanosar STZ at 200 mg/kg (group 2). The levels of glucose, insulin, and C-peptide when (a) fasting, (b) 30 minutes after eating, and (c) during intravenous glucose tolerance tests (IVGTTs) were measured in all pigs for 4 weeks after STZ injection. To confirm how long the diabetic state can be maintained after induction with STZ, levels were measured for 20 weeks in group 2. Results: One to 4 weeks after STZ administration, in group 1 (150 mg/kg) pigs, insulin and C-peptide levels were detected up to 7 &mgr;IU/mL and 0.4 ng/mL, respectively, both when fasting and after a meal test or IVGTT, indicating that the pigs had failed to become fully diabetic. In group 2 (200 mg/kg) pigs, insulin and C-peptide levels were less than the 2 &mgr;IU/mL and 0.25 ng/mL respective detection levels and did not increase after a meal test or IVGTT. Group 2 remained completely diabetic for the entire 20-week period of follow-up, without STZ-related hepatic or renal dysfunction. Conclusions: High-dose (200 mg/kg) Zanosar STZ induces diabetes safely and completely in pigs without side effects. Pancreatectomy can, therefore, be avoided.Abbreviations: ALT - alanine aminotransferase, AST - aspartate aminotransferase, AUC - area under the curve, iv - intravenous, IVGTT - intravenous glucose tolerance test, STZ - streptozotocin, Tx - allotransplantation

An in vitro model of pig liver xenotransplantation—pig complement is associated with reduced lysis of wild-type and genetically modified pig cells

Hara H, Campanile N, Tai H‐C, Long C, Ekser B, Yeh P, Welchons D, Ezzelarab M, Ayares D, Cooper DKC. An in vitro model of pig liver xenotransplantation—pig complement is associated with reduced lysis of wild‐type and genetically modified pig cells. Xenotransplantation 2010; 17: 370–378.

Factors affecting the mortality of necrotizing fasciitis involving the upper extremities

PurposeNecrotizing fasciitis involving the upper extremities is an uncommon, but potentially life-threatening infection. Surgical records were reviewed to identify its mortality risk factors.MethodsA 10-year retrospective review was conducted of all patients with upper limb necrotizing fasciitis treated in a tertiary care hospital in northern Taiwan. The demographic data, physical and laboratory findings and salient information with respect to the treatment and clinical outcome were collected and analyzed.ResultsFourteen patients were identified. The mean age of the all-male cohort was 60.2 years (range, 44–83 years). Five of the patients died, yielding a mortality rate of 35.7%. At the time of presentation, all 14 patients had pain and swelling, but fever occurred in only 7 patients. Associated chronic debilitating diseases, of which diabetes mellitus was the most common, were present in 9 individuals. Patients underwent an average of two surgical debridements. The initial presentation in a state of altered consciousness or respiratory distress was found to be a statistically significant factors for eventual mortality (P < 0.05).ConclusionThe results showed that necrotizing fasciitis of the upper extremity is associated with a high mortality rate. Early diagnosis and referral for aggressive surgical treatment before the development of systemic toxic signs are therefore considered to be essential for survival.

Reduction of human-to-pig cellular response by alteration of porcine MHC with human HLA DPW0401 exogenes.

BACKGROUND In pig-to-human discordant xenotransplantation, the xenograft can be rejected by a formidable human xenogenic T-cell response, even if the graft has gone through hyperacute rejection or delayed xenograft rejection (acute vascular rejection). We therefore examined, in this study, whether the human-to-pig cellular response could be attenuated through the generation of a transgenic pig for human HLA II. METHODS With the technique of microinjection, we produced the HLA DPw0401 transgenic pig. The expression of the HLA DPw0401 gene on peripheral blood mononuclear cells (PBMCs) of the transgenic pig was examined by reverse transcriptase-polymerase chain reaction and flow cytometry. The antigenicity of the transgenic HLA DPw0401 molecule was tested by the HLA DPw0401-primed lymphocyte test reagent. The cellular response was analyzed by xenogenic mixed lymphocyte culture. RESULTS The mRNA and protein of HLA DPw0401 were expressed in the PBMCs of the transgenic pig. The PBMCs of the HLA transgenic pig induced a stronger cellular reaction to HLA DPw0401-primed lymphocyte test reagents than the nontransgenic littermate pig (n=7, P<0.01). In direct xenogenic mixed lymphocyte culture with responders from HLA DPw0401(+) humans, the PBMCs from the HLA DPw0401 transgenic pig, as compared with those from the normal pig, induced a lower degree of xenogenic cellular response to human PBMCs (n=4, P=0.08). CONCLUSIONS Our preliminary data demonstrated the possibility that the human HLA DPw0401 phenotype can be transferred onto porcine cells through the generation of HLA transgenic pigs and make the PBMCs of humans more tolerant to porcine cells.

The pig-to-primate immune response: relevance for xenotransplantation.

Abstract: Background: The allotransplantation of some solid organs can be associated with a graft‐vs.‐host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig‐to‐primate organ xenotransplantation.