Expression Analysis of Same-Patient Metachronous and Synchronous Upper Tract and Bladder Urothelial Carcinoma.

PURPOSE: We compared upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BUC) in same-patient metachronous UTUC and synchronous UTUC and BUC using next-generation sequencing. MATERIALS AND METHODS: Consecutive untreated same-patient samples of UTUC and BUC were macrodissected from unstained formalin-fixed, paraffin-embedded slides after quality control. Samples were divided into 4 groups: 1) UTUC-metachronous BUC, 2) BUC-metachronous UTUC, 3) synchronous UTUC-BUC, 4) UTUC without BUC. Exclusions were inadequate clinical data or histological tumor purity <30%. Whole transcriptome RNA sequencing was performed. After quality assessment, gene expression clusters using unsupervised hierarchical consensus clustering and correlation with pertinent clinicopathologic variables, a prior RNASeq data set and other published data were performed. RESULTS: RNAseq was performed on 95 samples (UTUC=61, BUC=34) from 40 untreated patients. Unsupervised consensus clustering segregated the tumors into 2 clusters that were enriched with BASE47 basal-like or luminal-like gene expression. Almost two-thirds (61.9%) of Group 2 tumors were basal-like, while the majority of Groups 1, 3, 4 (80.6%, 70.0% and 69.6%, respectively) were luminal-like (p=0.017). Further analyses revealed that the differences in basal-like and luminal-like gene expression were associated with differential fibroblast and immune cell gene expression signatures. In all, 87.5% of metachronous tumors maintained subtype membership. CONCLUSIONS: Gene expression analysis of same-patient metachronous UTUC-BUC suggests that the majority of mUTUC developing after BUC appear more basal-like, while synchronous and initial UTUC tumors appear luminal-like. Metachronous tumors largely maintain molecular subtype membership of the initial tumor regardless of chronologic development or anatomical origin.

Xanthogranulomatous Ureteritis Mimicking Ureteral Involvement by Cancer in a Radical Cystectomy Specimen.

Xanthogranulomatous pyelonephritis is well established as a renal mass-forming inflammatory process. However, a ureteral counterpart is minimally recognized. In this article, we present a case of xanthogranulomatous ureteritis in an 81-year-old woman, mimicking ureteral involvement by cancer in a radical cystectomy specimen for invasive urothelial carcinoma. Similar to the pathogenesis of xanthogranulomatous pyelonephritis, the patient was noted to have ureteral obstruction by calculus and had urine culture positive for Klebsiella pneumoniae. To our knowledge, this is the first report of xanthogranulomatous ureteritis associated with this pathogen and the only report associated with concurrent bladder cancer. Increased pathologist and urologist awareness of xanthogranulomatous ureteritis expands the spectrum of pseudotumoral processes of the ureter.

Determination of urinary prostaglandin E2 as a potential biomarker of ureteral stent associated inflammation.

Ureteral stents are the most widely used surgical implant in urology. However, they may cause adverse effects to patients, including pain, discomfort, and inflammation. In this work, the inflammatory effect of stent placement and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed. Furthermore, a capillary electrophoresis mass spectrometry (CE-MS) based approach was subsequently developed to quantify urinary prostaglandin E2 (PGE2), a COX-2 metabolite known to contribute to inflammatory renal diseases, to further interrogate the role of this pathway. Urine samples were cleaned and preconcentrated by solid-phase extraction (SPE), and an on-line sample stacking method was used for the enrichment of analytes. The accuracy, precision, and specificity of this method were validated. Standard addition methods were performed to assess the reliability of using deuterated internal standards (IS) in compensating the remaining matrix effect after SPE as well as the detector fluctuation. Through the analysis of 32 pig urine samples, a statistically significant increase of PGE2 was observed in the stented group compared to the unstented (P = 0.01) and the recovered (P = 0.004) groups. This work determined that stent placement may contribute to COX-2-dependent inflammation and developed a reliable CE-MS based methodology to quantify PGE2 in stented individuals that may further understand the biology of stent-associated inflammation and inform urologic patient management.

Association of cancer progression with elevated expression of programmed cell death protein 1 ligand 1 by upper tract urothelial carcinoma and increased tumor-infiltrating lymphocyte density.

BACKGROUND: Increased expression of programmed cell death 1 ligand 1 (PD-L1) by tumor cells is thought to be a mechanism through which solid cancers promote immune tolerance. However, the association between PD-L1 expression and the prognosis of upper urinary tract urothelial carcinoma (UTUC) remains unknown. METHODS: We examined immunohistochemical PD-L1 expression and the tumor-infiltrating lymphocyte density (TILD) in 79 patients with UTUC who underwent nephroureterectomy. We classified the tumors into four types based on the combination of PD-L1 expression and TILD, and studied the clinicopathological characteristics of these four tumor types. RESULTS: Elevated expression of PD-L1 by tumor cells and a higher TILD were associated with a worse histological grade, higher pT stage, and higher peripheral blood neutrophil-to-lymphocyte ratio. Elevated expression of PD-L1 by tumor cells, a higher TILD, and type I, III, or IV tumors with elevated expression of either PD-L1 or TILD showed a positive correlation with poorer differentiation and local invasion. These three variables were associated with shorter progression-free survival and overall survival in univariate analysis, but only the latter was an independent determinant according to multivariate analysis. The patients who had type II tumors with lower PD-L1 expression and a lower TILD showed more favorable survival than the other three groups. CONCLUSIONS: These findings suggest that PD-L1 expression and TILs in the tumor microenvironment influence the progression of UTUC. Accordingly, it is important to understand the immunologic characteristics of the tumor microenvironment to develop more effective treatment strategies for this cancer.

A Rare Case of Extensive Unilateral Ureteritis Cystica.

Ureteritis cystica is rare, benign entity that associates with chronic urothelial irritation such as recurrent urinary tract infection or nephrolithiasis. It is often diagnosed incidentally on routine imaging or ureteroscopy in asymptomatic individuals. In this case report, we present the retrograde pyelogram and ureteroscopy images of a rare case of extensive unilateral ureteritis cystica in a 78-year-old female presenting for elective stone surgery.

THE MORPHOLOGICAL PICTURE OF LOCAL IMMUNE RESPONSES IN THE KIDNEYS, URETERS AND BLADDER OF THE FOETUSES AND NEWBORNS, WHO DEVELOPED IN CONDITIONS OF MATERNAL PREECLAMPSIA.

The purpose of the research consisted in revealing morphological peculiarities of local immune responses in the kidneys, ureters and bladder of the foetuses and newborns, who developed in conditions of maternal preeclampsia with different degrees of its severity. The research was conducted on autopsy material: the kidneys, ureters and bladders of mature foetuses and newborns. Four groups were formed in the study: group I - foetuses and newborns from mothers with physiological pregnancy; group II - foetuses and newborns from mothers, whose pregnancy was complicated with a mild degree of preeclampsia; group III - foetuses and newborns from mothers, whose pregnancy was complicated with a moderately severe degree of preeclampsia; group IV - foetuses and newborns from mothers, whose pregnancy was complicated with severe preeclampsia. An immune infiltration in the organs of the urinary system of foetuses and newborns was studied with help of histological, immunohistochemical and morphometric methods of examination. In the course of the study it was revealed that local immune responses in the kidneys, ureters and bladder of the foetuses and newborns passed with some abnormal deviations characterized by quantitative changes of СD3, СD4, СD8, СD20 and СD68 cells, whose degree of manifestation increased from the foetus to the newborn and with an aggravation of maternal preeclampsia. Mild, moderately severe as well as severe preeclampsia resulted in an extreme activation of the macrophage system, T cell and B cell immunity in the kidneys, ureters and bladder of foetuses and newborns that manifested itself with an increase of the absolute count of CD3, CD20 and CD68 cells. Mild preeclampsia caused an increase of the absolute count of CD8 cells and a decrease of the absolute count of CD4 cells with a resultant lowering of the immunoregulatory index. Moderately severe and severe preeclampsia caused an increase of the absolute count of CD4 cells and a decrease of the absolute count of CD8 cells with a corresponding elevation of the immunoregulatory index. In foetuses and newborns from mothers with physiological pregnancy as well as from mothers, whose pregnancy was complicated with preeclampsia having different degrees of severity, their kidneys were characterized by a more marked infiltration with immune cells versus their ureters and bladder. Thus, as a result of an excessive antigenic stimulation caused by maternal preeclampsia the local immune responses in the organs of the urinary system, which in physiological conditions fulfill exclusively protective functions, can become alterative and develop morphofunctional changes in the kidneys, ureters and bladder of foetuses and newborns.

The role of macrophages in the differentiation process of ureteral polyps.

Objective To evaluate the role of macrophage infiltration in the differentiation process of ureteral polyps and cancers. Methods This retrospective immunohistochemical study analysed archival samples of pathologically-confirmed specimens of low- and high-grade ureteral cancer, ureteral papilloma and ureteral polyps. The samples were immunohistochemically stained for cluster of differentiation (CD)4, CD8, CD16, CD25, CD56 and CD68 using immunofluorescence in order to identify different T-lymphocyte populations and macrophages. Results A total of 70 specimens were included in the analysis: 21 specimens of ureteral cancer, 17 specimens of ureteral papilloma, and 32 specimens of ureteral polyps. The largest proportion of CD4+CD25+ regulatory T cells was observed in the low-grade ureteral cancer group and almost none were observed in ureteral papillomas. The largest proportion of CD8+ cytotoxic T-lymphocytes was observed in the ureteral polyps. The largest proportion of CD56+ natural killer cells was detected in the ureteral polyps, with very low levels observed in the other three groups. The largest proportion of CD16+CD68+ macrophages was observed in the high-grade ureteral cancer group, which was significantly higher than that observed in the ureteral papillomas. Conclusions This study revealed that CD16+CD68+ macrophages appear to participate in ureteral neoplastic transformation.

Sorafenib Inhibits Renal Fibrosis Induced by Unilateral Ureteral Obstruction via Inhibition of Macrophage Infiltration.

AIMS: Sorafenib, which has been used extensively for the treatment of renal cell cancer and advanced hepatocellular carcinoma (HCC), has also been shown to have antifibrotic effects in liver fibrosis. However, the effects of sorafenib on renal fibrosis are unknown. Therefore, we investigated whether sorafenib inhibited renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and further explored the potential mechanism. METHODS: Mice underwent UUO followed by vehicle or sorafenib treatment. The expression of CD68, a macrophage marker, and the pro-inflammatory cytokines, MCP1 and CXCR3, were immunohistochemically analyzed. The involvement of macrophages in the formation of renal fibrosis was studied using confocal microscopy. RESULTS: Renal histopathology improved in the UUO-sorafenib mice. Sorafenib notably suppressed TGF-ß1-mediated renal fibrogenic effects. The mRNA and protein expressions of CD68, MCP1, and CXCR3 in the obstructed kidney were significantly decreased by sorafenib. Immunohistochemistry showed that CD68 and CXCR3 had a similar distribution, whereas MCP1 was observed predominantly in the tubular epithelial cells. Double immunofluorescence demonstrated that CD68-positive macrophages could co-localize with CXCR3. It also revealed that CXCR3 interacted with CXCL11 in the UUO mouse kidneys. Widespread adhesion of macrophages to myofibroblasts was markedly inhibited in UUO-sorafenib mouse kidneys. CONCLUSIONS: Taken together, the results indicated that sorafenib had protective effects against renal fibrosis; its mechanism of action was associated with inhibition of macrophage infiltration via the CXCR3/CXCL11 pathway. These data suggest the clinical potential of sorafenib for treatment of renal fibrosis and illustrate the immunological mechanisms underlying the protective effects of sorafenib.

Tolerance of bacteriuria after urinary diversion is linked to antimicrobial peptide activity.

OBJECTIVES: To compare the cationic antimicrobial peptide gene expression profiles and urinary cationic antimicrobial activities of patients after urinary diversion according to their urinary tract infection (UTI) status. Ileal conduit urinary diversion joins the bacterial-tolerant ileal epithelium and intolerant urothelium. After this procedure, one quarter of patients develop repeated symptomatic UTIs. Such development might reflect the altered innate immune mechanisms centered on epithelial expression and urinary activity of cationic antimicrobial peptides, such as defensins. METHODS: Ileal and ureteral biopsy specimens from ileal conduit subjects with (n = 18) and without (n = 18) recurrent symptomatic UTIs were assessed for cationic antimicrobial peptide gene expression using quantitative reverse transcriptase polymerase chain reaction. Overnight urine collections were analyzed for antimicrobial activity against a laboratory Escherichia coli strain, and infecting organisms were isolated from individual subjects. RESULTS: Overall, the ureteral epithelium showed increased expression of human α-defensin 5 and decreased expression of the human ß-defensin 1 after urinary diversion (P < .05). No significant changes were seen for the ileal epithelium. The expression levels of both defensins also did not differ significantly according to UTI status. Urinary cationic activity against infecting bacterial isolates from the individual subjects was significantly greater in those with symptomatic UTI (P < .001), and the activities against the laboratory E. coli strain were similar. CONCLUSIONS: The changes in the human ß-defensin 1 and human α-defensin 5 expression profiles and the link between symptomatic infection and high urinary antimicrobial activity suggest that innate mechanisms play significant roles in balancing bacterial tolerance and killing after ileal conduit urinary diversion. Future work needs to determine whether these changes can be therapeutically modulated to benefit the patients.

Interleukin-8 is essential for normal urothelial cell survival.

Interleukin-8 (IL-8; CXCL8) has been shown to play a role in multiple cellular processes. Here, we report an additional role of IL-8 as a growth and essential survival factor for normal human urothelial cells. Supplementing exogenous recombinant human IL-8 to normal urothelial cells promoted cell growth through the Akt pathway. Inhibition of IL-8 expression by small inhibitory RNA (siRNA) caused normal urothelial cells to die. Addition of recombinant human IL-8 rescued the normal urothelial cells treated with IL-8 siRNA. This rescue effect could be blocked by antibodies to the IL-8 receptor CXCR1 but not by CXCR2, suggesting that normal urothelial cells normally have IL-8 autocrine or paracrine activity for survival and growth mediated by CXCR1. IL-8 mRNA levels were lower in samples from patients with interstitial cystitis, a urinary bladder disorder associated with urothelial cell dysfunction and/or loss. Taken together, these results suggest that IL-8 is an important normal urothelial growth factor and is necessary for normal urothelial cell survival in vitro and in vivo. Lower IL-8 expression levels in the urinary bladder may contribute to pathophysiology of interstitial cystitis.

Dextranomer/hyaluronic Acid copolymer implant for vesicoureteral reflux: role of myofibroblast differentiation.

PURPOSE: Dextranomer/hyaluronic acid implantation is associated with a granulomatous inflammatory reaction, replaced by fibrosis. Appearance of myofibroblasts is considered a crucial event in fibrosis, and CD68 positive cells and other factors are implied in their activation. Mast cells are a source of these factors and tryptase can induce fibroblast to express alpha-smooth muscle actin, which is characteristic of myofibroblasts. We evaluated histological changes in refluxing ureters treated with dextranomer/hyaluronic acid and immunolocalized CD68 positive cells, tryptase mast cells and myofibroblasts. MATERIALS AND METHODS: We performed histological, histochemical and immunohistochemical analyses in 22 refluxing ureters treated with dextranomer/hyaluronic acid in comparison with 17 refluxing ureters who underwent ureteral reimplantation but did not receive endoscopic bulking agent. We used CD68 antibody for monocytes/macrophages and epithelioid cells, mast cell tryptase mouse antibody for mast cells, and alpha-smooth muscle actin and vimentin antibodies for myofibroblasts. The area of the ureteral lumen in dextranomer/hyaluronic acid treated and untreated ureteral endings was measured. RESULTS: Sirius red documented a major grade of histological lesions in dextranomer/hyaluronic acid treated refluxing ureters. CD68 and tryptase mast cell staining showed a significant enhancement of positive cells in dextranomer/hyaluronic acid treated refluxing ureters. Immunostaining for alpha-smooth muscle actin and vimentin displayed a myofibroblastic invasion in dextranomer/hyaluronic acid. Measurement of surface in treated refluxing ureters was significantly less than in untreated refluxing ureters. CONCLUSIONS: Our data documented a recruitment of CD68 and tryptase positive cells, abnormal accumulation of collagenous stroma and successive extracellular matrix remodeling through differentiation of myofibroblasts. Myofibroblasts might provoke tissue contraction, decreasing the ureteral diameter and modifying the ureteral length-to-diameter ratio, preventing urine reflux.

Decellularized ureter for tissue-engineered small-caliber vascular graft.

Previous attempts to create small-caliber vascular prostheses have been limited. The aim of this study was to generate tissue-engineered small-diameter vascular grafts using decellularized ureters (DUs). Canine ureters were decellularized using one of four different chemical agents [Triton-X 100 (Tx), deoxycholate (DCA), trypsin, or sodium dodecyl sulfate (SDS)] and the histology, residual DNA contents, and immunogenicity of the resulting DUs were compared. The mechanical properties of the DUs were evaluated in terms of water permeability, burst strength, tensile strength, and compliance. Cultured canine endothelial cells (ECs) and myofibroblasts were seeded onto DUs and evaluated histologically. Canine carotid arteries were replaced with the EC-seeded DUs (n = 4). As controls, nonseeded DUs (n = 5) and PTFE prostheses (n = 4) were also used to replace carotid arteries. The degree of decellularization and the maintenance of the matrix were best in the Tx-treated DUs. Tx-treated and DCA-treated DUs had lower remnant DNA contents and immunogenicity than the others. The burst strength of the DUs was more than 500 mmHg and the maximum tensile strength of the DUs was not different to that of native ureters. DU compliance was similar to that of native carotid artery. The cell seeding test resulted in monolayered ECs and multilayered alpha-smooth muscle actin-positive cells on the DUs. The animal implantation model showed that the EC-seeded DUs were patent for at least 6 months after the operation, whereas the nonseeded DUs and PTFE grafts become occluded within a week. These results suggest that tissue-engineered DUs may be a potential alternative conduit for bypass surgery.

Idiopathic retroperitoneal fibrosis: clinicopathologic features and differential diagnosis.

Idiopathic retroperitoneal fibrosis (IRF) is a rare disease often causing obstructive uropathy. We evaluated the clinicopathologic features of 24 patients with IRF to characterize the histopathology of the disease and to provide a framework for the differential diagnosis with other retroperitoneal fibrosing conditions. Retroperitoneal specimens were analyzed by light and electron microscopy and by immunohistochemistry. Most patients presented with abdominal/lumbar pain, constitutional symptoms, and high acute-phase reactants. Overall, 20 had ureteral involvement and 13 developed acute renal failure. The retroperitoneal tissue consisted of a fibrous component and a chronic inflammatory infiltrate with the former characterized by myofibroblasts within a type-I collagen matrix. The infiltrate displayed perivascular and diffuse patterns containing lymphocytes, macrophages, plasma cells, and eosinophils. The perivascular aggregates had a central core of CD20(+) cells and a mantle of CD3(+) cells in equal proportions. In the areas of diffuse infiltrate, CD3(+) cells outnumbered the CD20(+) cells. Most plasma cells were positive for the IgG4 isotype. Small vessel vasculitis was found in the specimens of 11 patients. Our study indicates that a sclerotic background with myofibroblasts associated with a diffuse and perivascular infiltrate mainly consisting of T and B lymphocytes may be a pathological hallmark of IRF.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

Novel nuclear factor kappa B activation inhibitor prevents inflammatory injury in unilateral ureteral obstruction.

PURPOSE: We determined whether the novel nuclear factor kappa B activation inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which is derived from epoxyquinomicin C, affects renal inflammatory responses in unilateral ureteral obstruction. MATERIALS AND METHODS: DHMEQ (8 mg./kg.) was administered to rats 1 day after unilateral ureteral obstruction and every day thereafter. Kidneys were harvested at day 7 after unilateral ureteral obstruction. Tissue nuclear factor kappa B activity and transforming growth factor-beta were determined by electrophoretic mobility shift assay and bioassay using mink lung epithelial cells, respectively. Renal tubular proliferation and apoptosis were detected by immunostaining proliferating cellular nuclear antigen and the TUNEL (Intergen, Purchase, New York) assay, respectively. Leukocyte infiltration was detected by immunostaining for CD45. Fibrosis was assessed by measuring tissue hydroxyproline content. RESULTS: Unilateral ureteral obstruction for 7 days significantly activated nuclear factor kappa B, induced tubular apoptosis, proliferation and interstitial fibrosis in the obstructed kidney of the control group compared with their unobstructed counterparts (30.3 +/- 4.5 nuclei per high power field versus 1.7 +/- 0.4, 25.7 +/- 3.3 nuclei per high power field versus 3.2 +/- 0.4 and 6.2 +/- 0.3 micromol. hydroxyproline per gm. tissue versus 3.4 +/- 0.1, respectively). Conversely daily administration of DHMEQ (8 mg./kg.) significantly inhibited nuclear factor kappa B activation and decreased mean tubular apoptosis (9.5 +/- 2.1 nuclei per high power field), proliferation (10.2 +/- 2.4 nuclei per high power field) and interstitial fibrosis (4.9 +/- 0.4 micromol. hydroxyproline per gm. tissue) in the obstructed kidney. CONCLUSIONS: Specific inhibition of nuclear factor kappa B can prevent inflammatory renal responses, suggesting that targeting nuclear factor kappa B activation may be feasible for preventing inflammatory kidney diseases.

Mast cell activation triggers a urothelial inflammatory response mediated by tumor necrosis factor-alpha.

PURPOSE: Mast cells have been implicated in bladder inflammation and pathogenesis. To determine if mast cell secretion products can modulate urothelial inflammatory responses we developed an in vitro model of mast cell-urothelial cell interactions. MATERIALS AND METHODS: Cultures of the immortalized urothelial cell line TEU-2 were incubated in the conditioned medium of mast cell cultures. The urothelial inflammatory response to mast cell secretion products was then determined by quantifying nuclear factor kappaB activity, the expression of endogenous nuclear factor kappaB dependent genes and the protein expression of inflammation markers. RESULTS: Conditioned medium from RBL-2H3 mast cells induced a 4-fold increase in TEU-2 nuclear factor kappaB activity that was independent of the activation state of the mast cells. In contrast, ribonuclease protection assays revealed that the nuclear factor kappaB dependent transcripts tumor necrosis factor-alpha (TNF-alpha), interleukin (IL) 8 and 1beta, and intracellular adhesion molecule 1 (ICAM-1) were induced by mast cell conditioned medium in a manner that strictly depended on mast cell activation (antigen challenge of IgE sensitized RBL-2H3 cells). The dependence on mast cell activation was confirmed by the observation that IL-8 secretion and ICAM-1 protein expression in TEU-2 cultures were induced only by conditioned medium of stimulated RBL-2H3 cells The induction of TEU-2 IL-8 secretion and ICAM-1 expression by mast cell conditioned medium could be blocked by an anti-TNF-alpha antibody or the cysteine protease inhibitor N-acetyl-leucinyl-leucinyl-norleucinal. CONCLUSIONS: Our data support the hypothesis that mast cells may participate in bladder inflammation. Furthermore, TNF-alpha acting via the nuclear factor kappaB signaling pathway may be a mediator of the urothelial response to mast cell secretion products.

Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption.

BACKGROUND: Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model. METHODS: Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics. RESULTS: In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis. CONCLUSIONS: This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.