Mussismilia braziliensis White Plague Disease Is Characterized by an Affected Coral Immune System and Dysbiosis.

Infectious diseases are one of the major drivers of coral reef decline worldwide. White plague-like disease (WPL) is a widespread disease with a complex etiology that infects several coral species, including the Brazilian endemic species Mussismilia braziliensis. Gene expression profiles of healthy and WPL-affected M. braziliensis were analyzed in winter and summer seasons. The de novo assembly of the M. braziliensis transcriptome from healthy and white plague samples produced a reference transcriptome containing 119,088 transcripts. WPL-diseased samples were characterized by repression of immune system and cellular defense processes. Autophagy and cellular adhesion transcripts were also repressed in WPL samples, suggesting exhaustion of the coral host defenses. Seasonal variation leads to plasticity in transcription with upregulation of intracellular signal transduction, apoptosis regulation, and oocyte development in the summer. Analysis of the active bacterial rRNA indicated that Pantoea bacteria were more abundant in WPL corals, while Tistlia, Fulvivirga, and Gammaproteobacteria Ga0077536 were more abundant in healthy samples. Cyanobacteria proliferation was also observed in WPL, mostly in the winter. These results indicate a scenario of dysbiosis in WPL-affected M. braziliensis, with the loss of potentially symbiotic bacteria and proliferation of opportunistic microbes after the start of the infection process.

Ecogenomics and metabolic potential of the South Atlantic Ocean microbiome.

The unique combination of depth, salinity, and water masses make the South Atlantic Ocean an ecosystem of special relevance within the global ocean. Yet, the microbiome of this ecosystem has received less attention than other regions of the global Ocean. This has hampered our understanding of the diversity and metabolic potential of the microorganisms that dwell in this habitat. To fill this knowledge gap, we analyzed a collection of 31 metagenomes from the Atlantic Ocean that spanned the epipelagic, mesopelagic and bathypelagic zones (surface to 4000 m). Read-centric and gene-centric analysis revealed the unique taxonomic and functional composition of metagenomes from each depth zone, which was driven by differences in physical and chemical parameters. In parallel, a total of 40 metagenome-assembled genomes were obtained, which recovered one third of the total community. Phylogenomic reconstruction revealed that many of these genomes are derived from poorly characterized taxa of Bacteria and Archaea. Genomes derived from heterotrophic bacteria of the aphotic zone displayed a large apparatus of genes suited for the utilization of recalcitrant organic compounds such as cellulose, chitin and alkanes. In addition, we found genomic evidence suggesting that mixotrophic bacteria from the bathypelagic zone could perform carbon fixation through the Calvin-Benson-Bassham cycle, fueled by sulfur oxidation. Finally, we found that the viral communities shifted throughout the water column regarding their targeted hosts and virus-to-microbe ratio, in response to shifts in the composition and functioning their microbial counterparts. Our findings shed light on the microbial and viral drivers of important biogeochemical processes that take place in the South Atlantic Ocean.

Groundwater vulnerability and risk mapping using GIS, modeling and a fuzzy logic tool.

A groundwater vulnerability and risk mapping assessment, based on a source-pathway-receptor approach, is presented for an urban coastal aquifer in northeastern Brazil. A modified version of the DRASTIC methodology was used to map the intrinsic and specific groundwater vulnerability of a 292 km(2) study area. A fuzzy hierarchy methodology was adopted to evaluate the potential contaminant source index, including diffuse and point sources. Numerical modeling was performed for delineation of well capture zones, using MODFLOW and MODPATH. The integration of these elements provided the mechanism to assess groundwater pollution risks and identify areas that must be prioritized in terms of groundwater monitoring and restriction on use. A groundwater quality index based on nitrate and chloride concentrations was calculated, which had a positive correlation with the specific vulnerability index.

[Video-electroencephalography prolonged monitoring in patients with ambulatory diagnosis of medically refractory temporal lobe epilepsy: application of fuzzy logic's model]. / Monitorización prolongada por videoelectroencefalografía de pacientes con diagnóstico ambulatorio de epilepsia del lóbulo temporal de difícil control: aplicación del modelo de lógica fuzzy

INTRODUCTION: The video-electroencephalography (video-EEG) prolonged monitoring is an important auxiliary diagnostic instrument in epilepsy, and provides valuable information to classify the type of crisis and epileptic syndromes and to localize the epileptogenic zone. The fuzzy logic gives an efficient and intelligent analysis method, able to make inferences over ambiguous systems, and has been increasing used in several areas. AIMS: To correlate clinical and electroencephalographic data obtained with video-EEG of patients with clinical and interictal electroencephalography criteria of medically refractory temporal lobe epilepsy (TLE) and to make inferences of diagnosis precision with the application of the Fuzzy Hierarchy COPPE/Cosenza Model; to investigate the frequency in which the clinical, syndromic and topographic diagnosis can be modified in these patients; to evaluate the usefulness and applicability of fuzzy logic on the analysis of this type of study. PATIENTS AND METHODS: Transversal, prospective study, that included 22 adult outpatients with ambulatory diagnosis of medically refractory TLE, submitted to video-EEG monitoring that varied from 48 hours to 10 days. The grade of diagnosis precision was infered by fuzzy logic. RESULTS: The clinical diagnosis of epilepsy was modified in two (9%) patients, the syndromic in six (27.2%) and the topographic in 16 (72.7%). The fuzzy indicators of ambulatory diagnostic precision were: 0.91 to clinical diagnosis; 0.74 to syndromic diagnosis and 0.36 to topographic diagnosis, considering 1 the maximal precision diagnostic value (attributed to the video-EEG results). CONCLUSIONS: The video-EEG prolonged monitoring established the clinical, syndromic and topographic correct diagnosis in these patients; the fuzzy logic best defined a mathematical value of the correspondence between ambulatorial diagnosis of TLE and the video-EEG prolonged monitoring.

Long-term results of metallic stents for benign biliary strictures.

BACKGROUND: Historically, surgical correction has been the treatment of choice for benign biliary strictures (BBS). Self-expandable metallic stents (MSs) have been useful for inoperable malignant biliary strictures; however, their use for BBS is controversial and their natural history unknown. HYPOTHESIS: To test our hypothesis that MSs provide only short-term benefit, we examined the long-term outcome of MSs for the treatment of BBS. Our goal was to develop a rational approach for treating BBS. DATA EXTRACTION: Between July 1990 and December 1995, 15 patients had MSs placed for BBS and have been followed up for a mean of 86.3 months (range, 55-120 months). The mean age of the patients was 66.6 years and 12 were women. Stents were placed for surgical injury in 5 patients and underlying disease in 10 patients (lithiasis, 7; pancreatitis, 2; and primary sclerosing cholangitis, 1). One or more MSs (Gianturco-Rosch "Z" for 4 patients and Wallstents for 11 patients) were placed by percutaneous, endoscopic, or combined approaches. We considered patients to have a good clinical outcome if the stent remained patent, they required 2 or fewer invasive interventions, and they had no biliary dilation on subsequent imaging. DATA SYNTHESIS: Metallic stents were successfully placed in all 15 patients, and the mean patency rate was 30.6 months (range, 7-120 months). Five patients (33%) had a good clinical result with stent patency from 55 to 120 months. Ten patients (67%) required more than 2 radiologic and/or endoscopic procedures for recurrent cholangitis and/or obstruction (range, 7-120 months). Five of the 10 patients developed complete stent obstruction at 8, 9, 10, 15, and 120 months and underwent surgical removal of the stent and bilioenteric anastomosis. Four of these 5 patients had strictures from surgical injuries. The patient who had surgical removal 10 years after MS placement developed cholangiocarcinoma. CONCLUSIONS: Surgical repair remains the treatment of choice for BBS. Metallic stents should only be considered for poor surgical candidates, intrahepatic biliary strictures, or failed attempts at surgical repair. Most patients with MSs will develop recurrent cholangitis or stent obstruction and require intervention. Chronic inflammation and obstruction may predispose the patient to cholangiocarcinoma.

Current management of recurrent pyogenic cholangitis.

Recurrent pyogenic cholangitis (RPC) is a chronic disease with multiple exacerbations requiring repeated biliary dilatation and stone removal. Even after adequate biliary drainage, most patients will have progression of intrahepatic disease. Management of patients with RPC is a multidisciplinary challenge for endoscopists, interventional radiologists, and surgeons because of the frequency and inaccessibility of strictures and stones. Complete stone clearance at any one operation is difficult. Hepaticojejunostomy with a subcutaneous afferent limb is a safe and effective way to provide access to the biliary tree for the management of patients with RPC. In our experience, trans-stomal cholangioscopic stricture dilatation followed by stone removal remains the basis of therapy in patients with RPC. By diligent surveillance, we should be able to eliminate or decrease the number of stones and prevent cholangitis and its sequelae.

Metastasis of hepatocellular carcinoma to the right colon manifested by gastrointestinal bleeding.

An 82-year-old black woman with a history of hepatocellular carcinoma presented with gastrointestinal bleeding. Barium enema and fibrocolonoscopy revealed a 4-cm polypoid mass at the level of the ascending colon with evidence of active bleeding. Biopsies of the lesion proved it to be metastatic hepatocellular carcinoma. Exploratory laparotomy revealed no further dissemination of the tumor, and the patient underwent an ileocolectomy. The serosal side of the colonic lesion was free from tumor, and there was no peritoneal implantation, direct extension, or lymph node involvement. This case represents an extremely rare presentation of metastatic hepatocellular carcinoma.

Surgical management of biliary gallstone disease during pregnancy.

BACKGROUND: Biliopancreatic gallstone disorders (BPD) manifesting during pregnancy are relatively rare. The management of these conditions remains controversial. Although perioperative problems and fetal loss have been reported, recent publications have advocated an early surgical approach. PATIENTS AND METHODS: Thirty-two pregnant women underwent operation for BPD between January 1993 and December 1997. The mean age was 29 years and ranged from 18 to 41 years. RESULTS: Twelve patients underwent a laparoscopic cholecystectomy (LC), and 20 open cholecystectomies (OC), including two conversions from laparoscopic. Seven of the OC patients required additional open CBD exploration and intraoperative choledochoscopy for CBD stones. No maternal mortality was observed. A single fetal demise (3%) occurred for a patient with gallstone pancreatitis who underwent open cholecystectomy during her 14th week of gestation. CONCLUSIONS: Early involvement of the obstetric team, with preoperative and postoperative fetal monitoring, and adequate management of anesthetic and tocolytic agents make cholecystectomy a safe procedure at any stage of pregnancy.

Efficacy of tacrolimus as rescue therapy for chronic rejection in orthotopic liver transplantation: a report of the U.S. Multicenter Liver Study Group.

BACKGROUND: A study was performed by 17 different U.S. liver transplantation centers to determine the safety and efficacy of conversion from cyclosporine to tacrolimus for chronic allograft rejection. METHODS: Ninety-one patients were converted to tacrolimus a mean of 319 days after liver transplantation. The indication for conversion was ongoing chronic rejection confirmed by biochemical and histologic criteria. Patients were followed for a mean of 251 days until the end of the study. RESULTS: Sixty-four patients (70.3%) were alive with their initial hepatic allograft at the conclusion of the study period and were defined as the responder group. Twenty-seven patients (29.7%) failed to respond to treatment, and 20 of them required a second liver graft. The actuarial graft survival for the total patient group was 69.9% and 48.5% at 1 and 2 years, respectively. The actuarial patient survival at 1 and 2 years was 84.4% and 81.2%, respectively. Two significant positive prognostic factors were identified. Patients with a total bilirubin of < or = 10 mg/dl at the time of conversion had a significantly better graft and patient survival than patients converted with a total bilirubin > 10 mg/dl (P=0.00002 and P=0.00125, respectively). The time between liver transplantation and conversion also affected graft and patient survival. Patients converted to tacrolimus < or = 90 days after transplantation had a 1-year actuarial graft and patient survival of 51.9% and 65.9%, respectively, compared with 73.2% and 87.7% for those converted > 90 days after transplantation. The mean total bilirubin level for the responder group was 7.1 mg/dl at the time of conversion and decreased significantly to a mean of 3.4 mg/dl at the end of the study (P=0.0018). Thirteen patients (14.3%) died during the study. Sepsis was the major contributing cause of death in most of these patients. CONCLUSIONS: Our results suggest that conversion to tacrolimus for chronic rejection after orthotopic liver transplantation represents an effective therapeutic option. Conversion to tacrolimus before development of elevated total bilirubin levels showed a significant impact on long-term outcome.

Transjugular intrahepatic portosystemic shunt: efficacy for the treatment of portal hypertension and impact on liver transplantation.

Variceal bleeding (VB) and ascites refractory to diuretics (RA) represent a significant cause of morbidity and mortality in patients with portal hypertension. Transjugular intrahepatic portosystemic shunts (TIPS) have been used effectively in patients with these complications, especially those individuals awaiting orthotopic liver transplantation (OLT). From April 1992 to July 1995, 41 adult patients underwent an attempt at TIPS placement for refractory VB or ascites at Cedars-Sinai Medical Center. Technical success was achieved in 37 of 41 cases (90.3%) with only two technical complications. Immediate control of hemorrhage and significant improvement of ascites was obtained in 91.9% and 83.5% of the patients, respectively. Six patients (16.2%) died within a week of TIPS placement due to uncontrollable ascites and multiorgan failure. Four of 31 patients (12.9%) developed mild to moderate grades of hepatic encephalopathy that was controlled with lactulose. Rebleeding from recurrent portal hypertension was noted in 5 of 31 cases (16.1%). Shunt stenosis or occlusion was seen in 7 of 31 cases (22.6%) at an average of 6.3 months following TIPS placement. Six patients underwent OLT within an average of 87 days after TIPS. These results indicate that TIPS appears to be an effective method for treatment of refractory VB and RA, especially for patients who are poor candidates for a surgical shunt or awaiting OLT. However, TIPS may not be considered a definitive solution for all patients with portal hypertension because of its current rate of shunt occlusion or stenosis.

Hepatic resection for malignancy in the elderly.

Although elderly patients are accounted for in all large series of major hepatic resections, the role of age as a determinant of outcome remains unclear. At Cedars-Sinai Medical Center, we review a series of 20 major hepatectomies for neoplasia performed in patients older than 66 years of age (4 of them > or = 80 years old) over a 5-year period. A retrospective comparison was conducted with a group of 22 hepatectomies for malignancy performed in 20 patients younger than 59 years of age during the same time period. The younger group had a significantly greater degree of liver resected (12 trisegmentectomies vs 3). Although one operative death (5% mortality) was observed in the elderly group, no statistically significant difference was noted, when compared to the younger group (Chi-square, P = 0.48). Likewise, no significant difference in the complication rate (20% vs 33%) was noticed (Chi-square, P = 0.8). Severe postoperative liver dysfunction was present in 2 cases (10%) in the elderly group and one (4%) in the younger group. These patients underwent a right trisegmentectomy (TS). Nine patients from each group were resected without red blood cell transfusion. We conclude that major hepatic resection in elderly patients without severe comorbid disease is a safe procedure that is not associated with an increased perioperative morbidity or mortality rate.

The use of a pig liver xenograft for temporary support of a patient with fulminant hepatic failure.

A 26-year-old female patient with fulminant hepatic failure and a history of autoimmune hepatitis was heterotopically transplanted with a pig hepatic xenograft to provide temporary metabolic support prior to transplantation with a human donor organ. Circulating natural antipig antibodies were removed prior to transplantation by plasmapheresis and ex vivo en bloc perfusion of the donor pig kidneys. The liver xenograft functioned after transplantation as measured by active bile production, stabilization of prothrombin levels, and reduction in the circulating levels of lactic acid and the enzymes AST and ALT. Despite the removal of greater than 90% of the recipient's natural xenoantibodies prior to transplantation, the levels of antibody rapidly returned and were associated with antibody and complement-mediated rejection of the donor graft. Immunohistochemical evidence of graft rejection could be detected by the deposition of antibody, complement components including properdin, and endothelial swelling as early as 3 hr posttransplantation. These lesions progressed in severity and were accompanied by evidence of thrombosis and ischemic necrosis of the liver xenograft by 34 hrs posttransplantation. The main portal vein, hepatic artery, and vena cava were patent. The placement of the liver graft did not result in any improvement in the neurological status of the patient and she died 34 hr after xenografting due to irreversible brain damage. The information derived from this case has renewed interest in the clinical use of bioartificial devices and whole organ perfusion using xenogeneic tissue for temporary bridging of patients prior to allografting.

Intragraft cytokine gene expression in human liver allografts.

Cytokines are thought to play an important role in the inflammatory and immune responses of allograft rejection. We evaluated the pattern of cytokine gene expression in 36 liver biopsy specimens obtained from 20 recipients of primary orthotopic liver allografts. Specific mRNA expression was identified by a polymerase chain reaction (PCR) using oligonucleotide primers specific for human interleukin (IL)-1 beta, IL-2, IL-4, IL-6, IL-10 Interferon (IFN) gamma, tumor necrosis factor (TNF)-alpha and beta-actin. We detected IL-1 beta, IL-6 and IFN-gamma cytokine message most consistently in patients with rejecting liver allografts. TNF-alpha and IL-2 were also observed in rejecting livers, but only during the early phases of the reaction. IL-4 was expressed in the majority of liver allograft biopsy specimens, regardless of the presence or absence of clinical or pathological evidence of rejection. Sequential biopsy specimens in rejecting allografts showed decreased cytokine expression after the induction of a positive response to immunosuppressive therapy. The analysis of biopsy specimens from stable liver grafts showed a predominance in the expression of IL-10. These results may reflect a differential production of inflammatory and regulatory cytokines in response to liver allograft rejection in transplant recipients. They suggest that three cytokines, IL-1 beta, IL-6 and IFN-gamma, may play an important role as markers for liver allograft rejection. Conversely, IL-10 expression was noted in patients with stable graft function. This pattern of expression may correlate with host immune responses that allow for prolonged, rejection-free survival of the graft.

Combination therapy with Brequinar sodium and cyclosporine synergistically prolongs hamster-to-rat cardia xenograft survival.

The accelerated rejection of hamster cardiac xenografts by Lewis rat recipients is due primarily to a humoral immune response. Traditional immunosuppressive agents, including cyclosporine, FK 506, and rapamycin, have not been effective in prolonging the rejection of xenografts in this model. We have recently examined the ability of the combination of cyclosporine and a new immunosuppressive agent, Brequinar sodium, to prevent the rejection of hamster xenografts. Prolongation of xenograft survival by treatment with Brequinar sodium alone (3 mg/kg/day) was minimally effective, with a median survival of 5.5 days. Conversely, a combination of Brequinar sodium (3 mg/kg/day) and cyclosporine (10 mg/kg/day), was associated with a significant prolongation of median graft survival (> 100 days). A quantitative analysis of the dose-effect relationship of Brequinar sodium and cyclosporine demonstrated a potent synergistic interaction for this combination therapy (combination index < 1). A sharp increase occurred in the binding of immunoglobulin M antibodies at day 4 after transplantation in the serum of untreated and cyclosporine-treated Lewis recipients of cardiac xenografts, whereas a clear decrease occurred in anti-hamster immunoglobulin M antibody production during this period of time in the animals treated by combined immunosuppression. Inhibition of anti-donor immunoglobulin M antibody production, as evaluated by the direct enzyme-linked immunosorbent assay and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide measurements, extended for more than 100 days after transplantation in the combination therapy group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of orthotopic liver allograft rejection in rats with a short-term brequinar sodium therapy. Analysis of intragraft cytokine gene expression.

Brequinar sodium (BQR) is a new immunosuppressive drug that is highly effective in preventing graft rejection in several different experimental settings, including primary allografts and xenografts. A short course of BQR treatment during the onset of allograft rejection can induce the permanent survival of liver and kidney allografts in rats. To study the molecular basis of BQR-induced prolongation of allograft survival, we analyzed the intragraft pattern of IL-1 alpha, IL-2, IL-2R, IL-4, IL-6, IL-10, and TNF gene expression in the ACI-to-LEW liver allograft model. A semiquantitative polymerase chain reaction was developed to measure cytokine gene expression in control and BQR-treated liver graft recipients at various days after transplantation. Untreated control liver allografts expressed all of the cytokines analyzed. There was a marked increase in the steady state level of transcripts for each cytokine as graft rejection proceeded. The treatment of liver graft recipients with 12 mg/kg/day of BQR on days 6, 7, and 8 after transplantation suppressed the expression of all these cytokines within 24 hr of administration. The early suppression of cytokine expression was associated with a modest but distinct reduction in the infiltration of inflammatory cells into the liver grafts. The reduction in the level of transcripts for IL-4, IL-6, and IL-10 persisted in long-term survivors (30 days after transplantation). In contrast, there was a significant increase in the level of transcripts for IL-1 alpha, IL-2, and IL-2R in these long-term survivors. Our results demonstrated clearly that the pattern of cytokine gene expression during allograft rejection is significantly altered by a 3-day course of therapy with BQR. The temporary down-regulation of cytokine gene expression may be responsible for an altered immunological state that results in the prolonged survival of liver allografts.

Protective effect of the lazaroid U74006F in cold ischemia-reperfusion injury of the liver.

Lipid peroxidation may play a major role in the loss of liver graft viability after prolonged cold ischemia and reperfusion injury. The lazaroid compound U74006F is a potent inhibitor of lipid peroxidation, and this study was designed to evaluate the efficacy of this compound in preventing cold ischemia-reperfusion damage in three different models: pig endothelial cells in culture, ex vivo isolated pig liver perfusion and orthotopic transplantation of syngeneic rat livers. The addition of U74006F to University of Wisconsin preservation solution significantly prolonged endothelial cell viability after 48 and 72 hr of cold ischemia and reoxygenation (p < 0.01). Donor pigs were injected with vehicle or U74006F (4.5 mg/kg) before liver harvest. After 24 hr of cold storage in University of Wisconsin solution, the livers were perfused with pig blood for 180 min in an isolation chamber. Measurements of liver function parameters, including AST, ALT, bile production, superoxide anion and phospholipase A2 release, were assessed every 60 min. Although bile production was similar in the U74006F-treated and control groups, significant decreases of AST and ALT levels (p < 0.01) in the perfusate of the livers from treated donors were observed. In addition, the U74006F group displayed significantly reduced release of superoxide anion and phospholipase A2 compared with these parameters in the untreated group (p < 0.05 and p < 0.01, respectively). In the last model, donor rats were treated with U74006F before harvest; the rat liver grafts were preserved in cold University of Wisconsin solution for 24 hr and then transplanted into recipient rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The synergism of brequinar sodium and cyclosporine used in combination to prevent cardiac allograft rejection in the rat.

Brequinar sodium (BQR) is a novel immunosuppressive drug that inhibits cell proliferation by virtue of its disruption of the de novo pyrimidine biosynthesis. The basis of the immunosuppressive activity of BQR is distinctively different from that of cyclosporine (CsA), and we have recently evaluated in vivo and in vitro the efficacy of the two drugs when used in combination. Subtherapeutic doses of BQR and CsA were tested for their ability to prolong heterotopic cardiac allograft survival in the MHC- and non-MHC-mismatched ACI-->LEW rat strain combination. The graft survival data derived from these experiments were analyzed using the median-effect analysis to establish the immunosuppressive interaction of both drugs. The administration of BQR 3 mg/kg three times weekly or CsA 2.5 mg/kg daily moderately prolonged cardiac allograft survival, with a mean survival of 10 +/- 0.5 and 16 +/- 5.3 days, respectively. The use of the two drugs in combination with the same dose schedule exerted a synergistic effect on graft survival, prolonging the graft function to a mean of 31 +/- 5.7 days. Sera from animals treated with the two drugs displayed, when compared with single treatment groups (BQR 3 mg/kg and CsA 2.5 mg/kg), significantly (P < 0.01) increased in vitro inhibition of lymphocyte proliferation following stimulation with PHA. Finally, a clear correlation between the mean survival time and BQR plasma levels of animals treated with BQR alone or in combination with CsA was seen. Those treatment groups with BQR levels below 2 micrograms/ml (1.5 and 3.0 mg/kg/3x/week) had a mean graft survival of less than 10 days). In contrast, recipients treated with a combination of low doses of BQR and CsA displayed higher drug plasma levels (> 2 micrograms/ml) and longer mean graft survival times. These observations suggest that BQR and CsA may be highly effective when used in combination to prevent organ allograft rejection for clinical transplantation.

Pig aortic endothelial cell antigens recognized by human IgM natural antibodies.

Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (> 100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.

Removal of natural human xenoantibodies to pig vascular endothelium by perfusion of blood through pig kidneys and livers.

We have examined the nature of the binding of human xenoantibodies to pig liver and kidney vascular endothelium. Our results demonstrate that human serum contains IgM and IgG xenoantibodies that bind to pig vascular endothelium, and that the pattern of antibody binding is similar for both livers and kidneys. Immunohistochemical analysis of pig kidneys after perfusion with human blood demonstrated the binding of both IgM and IgG xenoantibodies, complement (C3), and fibrinogen to the vascular and glomerular endothelium. An ELISA assay of the perfusate after perfusion of 500 ml of human blood through a single pig kidney for 60 min demonstrated a significant reduction in the amount of human IgM (67%) and IgG (55%) binding to pig aortic endothelium. Similar perfusion experiments conducted with pig livers were associated with minimal immunohistochemical evidence of the binding of human xenoantibodies to liver vascular endothelium. Immunofluorescence staining for IgM, IgA, C3, and C1q was negative or minimally positive in the liver vascular endothelium. Sinusoidal endothelium were weakly positive for IgG and fibrinogen. The perfusion of the pig liver with human blood was, however, associated with a significant reduction in the subsequent binding of IgM and IgG to pig kidney vascular endothelium. Pig liver perfusion was also responsible for the removal of both IgM and IgG xenoantibodies capable of reacting with pig aortic endothelium, as measured by an ELISA assay of the perfusate. These results suggest that both pig kidney and livers are capable of absorbing the xenoantibodies that may be responsible for mediating a hyperacute rejection of pig xenografts and that the distribution of the target antigens for these antibodies is similar in the two organs.