Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa.
Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here.
Digital imaging; progenitor cells; Canals of Hering; hepatocyte nuclear factors; fluorescence microscopy
To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure.
Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol (“Pittsburgh protocol”).
Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring.
All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates.
Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.
bone marrow; cell therapy; composite tissue allotransplantation; hand transplantation; immunomodulation; immunomonitoring; immunosuppression; reconstructive transplantation; rejection; vascularized composite allotransplantation
Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. “-Omics” analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: a) spatial-temporal relationships; b) rare events/cells; c) complex structural context; and d) integration into a “systems” model. Nevertheless, except for immunostaining, no transformative advancements have “modernized” routine microscopy in over 100 years.
Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology - global “–omic” analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes.
Digital image; Fibrosis; Quantitative analysis; Allograft pathology; C4d; 3-dimensional imaging; Quantum dots
Successful solid organ transplantation is generally attributed to the increasingly precise ability of drugs to control rejection. However, it was recently shown that a few donor haematolymphoid cells can survive for decades in recipients of successful organ allografts, a phenomenon called microchimaerism. The association for decades of haematolymphoid chimaerism with allograft tolerance in experimental transplantation suggests that immunosuppressive drugs merely create a milieu that enables an allograft and its complement of passenger leucocytes to prime the recipient for graft acceptance.
Exploitation of this concept requires a fundamental shift in the classical view of passenger leucocytes only as initiators of rejection. Microchimaerism has taught us that solid organ transplantation involves the transfer of two donor organ systems to the recipient: the allograft parenchyma and the donor haematolymphoid system in the form of donor stem cells contained within the passenger leucocyte compartment. Each has the potential to integrate with the corresponding recipient system and carry out normal physiological functions, such as immunological self definition. Resistance to initial integration by mature T cells requires some form of immunosuppression, but maintenance of donor immune system function will depend on renewable supply of cells, which can be provided by engrafted progenitors. Successful clinical application will depend on the development of low morbidity methods to enhance engraftment of donor haemopoietic stem cells.
Induction with lymphocyte-depleting antibodies is routinely employed to prevent rejection but often skews T cells towards memory. It is not fully understood which memory and regulatory T cell subsets are most affected and how they relate to clinical outcomes.
We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after Alemtuzumab induction. 34 healthy subjects and 9 patients with acute cellular rejection (ACR) were also studied.
We found that Alemtuzumab caused protracted CD4>CD8 T lymphocyte deficiency, increased proportion of CD4+ memory T cells (TM), and decreased proportion of CD4+ regulatory T cells (TREG). Reactive patients exhibited higher proportions of CD4+ effector memory (TEM) and CD8+ terminally differentiated effector memory (TEMRA), with greater CD4+ TEM and CD8+ TEMRA to TREG ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8+ TEMRA. Mixed lymphocyte assays showed significantly lower T cell proliferation to donor than third party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8+ T cells.
Our findings provide evidence that T cell skewing towards effector memory may be associated with anti-graft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.
kidney transplantation; memory T cells; regulatory T cells; alemtuzumab
The efficacy of alloSCT is limited by graft-versus-host disease (GVHD). Host hematopoietic antigen presenting cells (APCs) are important initiators of GVHD making them logical targets for GVHD prevention. Conventional dendritic cells (cDCs) are key APCs for T cell responses in other models of T cell immunity and they are sufficient for GVHD induction. However, we report here in two polyclonal GVHD models in which host hematopoietic APCs are essential, that GVHD was not decreased when recipient cDCs were inducibly or constitutively deleted. Additional profound depletion of plasmacytoid DCs and B cells, with or without partial depletion of CD11b+ cells, also did not ameliorate GVHD. These data indicate that, in contrast to pathogen models, there is a surprising redundancy as to which host cells can initiate GVHD. Alternatively, very low numbers of targeted APCs were sufficient. We hypothesize the difference in APC requirements in pathogen and GVHD models relates to the availability of target antigens. In anti-pathogen responses specialized APCs are uniquely equipped to acquire and present exogenous antigens whereas in GVHD all host cells directly present alloantigens. These studies make it unlikely that reagent-based host APC depletion will prevent GVHD in the clinic.
Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts.
We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG−/−) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection.
Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG−/− mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell- dependent chronic rejection.
The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.
innate immunity; chronic rejection; NK cells; B-1 lymphocytes
Graft-vs.-host disease (GVHD) caused by donor T cells attacking recipient tissues is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (alloSCT). Studies have shown that effector memory T cells (TEM) do not cause GVHD but are capable of immune functions post-transplant, including graft-vs.-leukemia (GVL) effects, but the reasons for this are unclear. In mice, the TEM pool may have a less-diverse T cell receptor (TCR) repertoire than TN with fewer alloreactive clones. We therefore tested whether enhancing the alloreactivity of TEM would restore their ability to cause GVHD. In an MHC-matched system, alloreactive TEM were created by transferring GVHD effector cells into syngeneic recipients and allowing conversion to TEM. Upon retransfer to freshly transplanted recipients, these cells caused only mild GVHD. Similarly, in an MHC-mismatched system, TEM with a proven increased precursor frequency of alloreactive clones only caused limited GVHD. Nonetheless these same cells mounted strong in vitro alloresponses and caused rapid skin graft rejection. TEM created from CD4 cells that had undergone lymphopenia-induced proliferation also caused only mild GVHD. Our findings establish that conversion to TEM significantly reduces GVHD potency, even in cells with a substantially enhanced alloreactive repertoire.
Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LTx). B7-H1, recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7-H1 plays crucial roles during innate immune responses induced by hepatic I/R injury and tested this hypothesis in the mouse LTx model using B7-H1 KO liver grafts with 24 hr cold storage. Cold I/R injury in WT to WT LTx enhanced constitutive B7-H1 expression on dendritic cells and sinusoidal endothelial cells, and promptly induced B7-H1 on hepatocytes. When B7-H1 KO liver grafts were transplanted into WT recipients, serum ALT levels and graft necrosis were significantly higher than WT to WT LTx. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3+ T cells, in particular CD8+ T cells. B7-H1 KO grafts had significantly lower incidences of Annexin V+ CD8+ T cells, indicating the failure to delete infiltrating CD8+ T cells. To evaluate the relative contribution of parenchymal and bone marrow-derived cell (BMDC) B7-H1 expression, chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDC were generated and transplanted into WT recipients. Selective B7-H1 deficiency on parenchymal cells or BMDC resulted in similar levels of ALT and liver injury, suggesting that both parenchymal and BMDC B7-H1expression is involved in the control of liver damage. Human livers upregulated B7-H1 expression after LTx. Conclusion: The study demonstrates that graft tissue expression of B7-H1 plays critical roles in regulating inflammatory responses during LTx-induced hepatic I/R injury, and suggests that negative coregulatory signals may have an important function in hepatic innate immune responses.
apoptosis; T cells; hepatocyte; nonparenchymal cells; dendritic cells
Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.
Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.
Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.
Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T cell migration to vascularized allografts.
CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3−/− and wildtype B6 mice and co-transferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hours later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues.
We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype vs CXCR3−/− CD4 and CD8 memory T cells to allografts could be detected in either wildtype or aly/aly hosts. In the latter, wildtype and CXCR3−/−memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3−/− memory T cells.
CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking either CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell homing to transplanted organs may be distinct from those required for their migration to sites of infection.
T lymphocyte; chemokine receptor; transplantation; rejection
Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
Spontaneous orthotopic liver allograft acceptance associated with microchimerism in mice induces tolerance to subsequent skin or heart transplants from the donor but not third-party animals. Despite in vivo hyporesponsiveness, in vitro MLC and CTL assays showed continuing antidonor reactivity. Cells isolated from recipients’ spleens and grafted livers, when tested in MLC and CTL assays, were antidonor reactive out to 3 months to the same degree as splenocytes obtained from either naive or presensitized (with skin or heart) mice. Nevertheless, passive transfer of splenocytes or liver lymphocytes from liver tolerant mice, but not naive or sensitized donor strain mice, were able to prolong skin graft survival significantly in naive irradiated recipients. By using a strain combination in which the donor but not the recipient expressed the stimulatory endogenous super-Ag (Mlsf), it was possible to determine whether super-Ag-reactive T cells bearing Vβ5 and Vβ11 were deleted or anergic. Phenotypic analysis of cells isolated from recipients’ spleens and grafted livers (up to 90 days after transplant), when compared with naive animals, showed no significant difference in Vβ5 and Vβ11 TCR expression. Additionally, when these isolated spleen cells were tested for antibody-mediated stimulation, both anti-Vβ5 and Vβ11 TCR mAb led to marked proliferation of cells obtained from naive and liver-transplanted recipients, but as expected, proliferation was very low in cells from naive donors. These results suggest that liver transplantation induces donor-specific tolerance in vivo, which may not be reflected in in vitro proliferative and cytotoxicity assays (split tolerance). Furthermore, this tolerance does not seem to be induced by clonal deletion or anergy of minor-lymphocyte-stimulating-antigen-reactive T cells in the recipients.
Although the persistence of multilineage microchimerism in recipients of long-surviving organ transplants implies engraftment of migratory pluripotent donor stem cells, the ultimate localization in the recipient of these cells has not been determined in any species.
Progenitor cells were demonstrated in the bone marrow and nonparenchymal liver cells of naive rats and in Brown Norway (BN) recipients of Lewis (LEW) allografts by semiquantitative colony-forming unit in culture (CFU-C) assays. The LEW allografts of bone marrow cells (BMC) (2.5×108), orthotopic livers, or heterotopic hearts (abdominal site) were transplanted under a 2-week course of daily tacrolimus, with additional single doses on days 20 and 27. Donor CFU-C colonies were distinguished from recipient colonies in the allografts and recipient bone marrow with a donor-specific MHC class II monoclonal antibody. The proportions of donor and recipient colonies were estimated from a standard curve created by LEW and BN bone marrow mixtures of known concentrations.
After the BMC infusions, 5–10% of the CFU-C in the bone marrow of BN recipients were of the LEW phenotype at 14, 30, and 60 days after transplantation. At 100 days, however, donor CFU-C could no longer be found at this site. The pattern of LEW CFU-C in the bone marrow of BN liver recipients up to 60 days was similar to that in recipients of 2.5 × 108 BMC, although the donor colonies were only 1/20 to 1/200 as numerous. This was expected, because the progenitor cells in the passenger leukocytes of a single liver are equivalent to those in 1–5×106 BMC. Using a liquid CFU-C assay, donor progenitor cells were demonstrated among the nonparenchymal cells of liver allografts up to 100 days. In contrast, after heart transplantation, donor CFU-C could not be identified in the recipient bone marrow, even at 14 days.
Under effective immunosuppression, allogeneic hematopoietic progenitors compete effectively with host cells for initial engraftment in the bone marrow of noncytoablated recipients, but disappear from this location between 60 and 100 days after transplantation, coincident with the shift of donor leukocyte chimerism from the lymphoid to the nonlymphoid compartment that we previously have observed in this model. It is possible that the syngeneic parenchymal environment of the liver allografts constitutes a privileged site for persistent progenitor donor cells.
Tacrolimus (FK506) is an effective immunosuppressant for human heart transplantation, but information about its effects on cardiac allograft and nonallograft kidney and liver histopathologic study is limited.
We therefore reviewed 1145 endomyocardial biopsy specimens and eight autopsy results from 80 heart transplant recipients who received tacrolimus as baseline immunosuppression. These were compared with 619 endomyocardial biopsy specimens and four autopsy results from 51 patients treated with cyclosporine-based immunosuppression with lympholytic induction (CLI) by use of rabbit anti-thymocyte globulin. Twenty-one histologic features including the International Society for Heart and Lung Transplantation histopathologic grade were retrospectively assessed without knowledge of the treatment regimen. The lymphocyte growth index on biopsy specimens obtained from these patients was also compared.
In general, there were no qualitative differences in the histopathologic appearance of various allograft syndromes between tacrolimus- and CLI-treated patients. Thus histopathologic criteria used to diagnose various graft syndromes are applicable under tacrolimus immunosuppression. However, early (between 10 and 30 days) after transplantation, biopsy specimens from patients treated with tacrolimus showed a significantly higher percentage of inflamed fragments (p = 0.02), the inflammation tended to be more severe (p = 0.09), and the rejection grade tended to be slightly higher (p = 0.08). In contrast, during the late transplantation period (275 to 548 days), biopsy specimens from patients treated with CLI showed a significantly higher percentage of inflamed fragments (p = 0.03), more severe inflammation (p = 0.03), higher rejection grades (p = 0.01), and a higher frequency of Quilty lesions (p = 0.05). Although overall freedom from any grade 3A or higher rejection was greater in the CLI-treated arm, tacrolimus was successfully used to treat refractory rejection in three patients from the CLI-treated arm. Concern has been raised in the literature about the possibility of tacrolimus being a direct hepatotoxin and an accelerant of allograft obliterative arteriopathy. However, no evidence to support either of these contentions was detected in this patient population. In contrast, tacrolimus is clearly nephrotoxic, although similar to cyclosporine in this regard.
Tacrolimus is an effective immunosuppressive drug for heart transplantation. The cardiac allograft histopathologic study of patients treated with tacrolimus immunosuppression does not significantly differ from those given conventional, cyclosporine-based triple therapy with lympholytic induction.
Recipient antigen presenting cells (APCs) are required for CD8-mediated GVHD and have an important and nonredundant role in CD4-mediated GVHD in mouse MHC-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs — dendritic cells, macrophages, and B cells — are not well defined. If recipient B cells are important APCs they could be depleted with Rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses and consequently B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient due to prior Rituximab. We therefore studied the role of recipient B cells in MHC-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4-and CD8-dependent models, B cell deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.
The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.
After a short course of tacrolimus, Lewis rat liver allografts induce donor-specific nonreactivity in Brown Norway recipients that is immunosuppression-independent after 28 days. To clarify the role of donor major histocompatibility complex (MHC) class II+ cells, we investigated the migration to the recipient splenic T- and B-cell compartments of different subsets of Lewis MHC class II+ passenger leukocytes. The rise and decline of immune activation were monitored in the hepatic allograft and in the host spleen by analyses of BrdU+ (proliferating) leukocytes, TUNEL+ (apoptotic) cells, apoptosis-associated molecules, TH1/ TH2 cytokine profiles, and histoimmunocytochemical examination of graft and splenic tissues. Serial flow cytometry studies during the 28-day period of drug-assisted “hepatic tolerogenesis” showed that migratory MHC class II+ cells accounted for less than half of the donor cells in the host spleen. The class II+ cells consisted mostly of B cells that homed to splenic B-cell follicles with only a sparse representation of dendritic cells that were exclusively found in the splenic periarteriolar lymphoid sheath. In parallel studies, transplantation of the less tolerogenic heart produced a diminutive version of the same events, but with far fewer donor cells in the host spleen, evidence of sustained immune activation, and the development of chronic rejection by 100 days. The data are consistent with the paradigm that migration of donor leukocytes is the prime determinant of variable tolerance induction induced by transplantation of the liver and other organs, but without regard for donor MHC class II+ expression.
Hamster hearts transplanted into stable rat recipients of hamster livers (OLT rats) were hyperacutely rejected after transfer with unaltered rat antihamster hyperimmune serum (HS). This was followed by immediate liver xenograft rejection in 4 of 5 rats. In contrast, simple heat inactivation of the rat HS resulted in prolonged survival of hamster hearts to 25 days without deterioration effect in the liver xenografts. This effect was species-specific because third-party mouse heart grafts in OLT rats were hyperacutely rejected in minutes if either active or heat inactivated antimouse HS was given. In cytotoxicity experiments, the complement in OLT serum produced weak lysis of hamster lymphocytes, while efficiently doing so with mouse cell targets. Because normal hamster serum caused no lysis at all of hamster target cells, the residual low-grade lysis of OLT serum was possibly being mediated by extrahepatic sources of rat C. In conclusion, the homology of C and target cells represents a mechanism of protection that the liver confers to other organs, and that is most easily seen in xenografts but may be allospecifically operational with allografts as well within the limits of MHC restriction.
Hepatocellular carcinoma often develops in the setting of abnormal hepatocyte growth associated with chronic hepatitis and liver cirrhosis. Transforming growth factor-βs (TGF-βs) are multifunctional cytokines pivotal in the regulation of hepatic cell growth, differentiation, migration, extracellular matrix production, stem cell homeostasis and hepatocarcinogenesis. However, the mechanisms by which TGF-βs influence hepatic cell functions remain incompletely defined. We report herein that TGF-β regulates the growth of primary and transformed hepatocytes through concurrent activation of Smad and phosphorylation of cPLA2α, a rate-limiting key enzyme that releases arachidonic acid for production of bioactive eicosanoids. The interplays between TGF-β and cPLA2α signaling pathways were examined in rat primary hepatocytes, human hepatocellular carcinoma cells and hepatocytes isolated from the newly developed cPLA2α transgenic mice. Our data show that cPLA2α activates PPAR-γ and thus counteracts Smad2/3-mediated inhibition of cell growth. Therefore, regulation of TGF-β signaling by cPLA2α and PPAR-γ may represent an important mechanism for control of hepatic cell growth and hepatocarcinogenesis.
Transforming growth factor-β; cytosolic phospholipase A2α; peroxisome proliferator activated receptor-γ; hepatocyte; liver
Primary biliary cirrhosis is a frequent indication for liver transplantation. The purpose of this report is to present our experience with liver transplantation for primary biliary cirrhosis. Attention is given to the causes of hepatic dysfunction seen in allografts. In addition, we review the postoperative problems encountered and the quality of life at time of last follow-up in patients with transplants for primary biliary cirrhosis. A total of 97 orthotopic liver transplant procedures were performed in 76 patients with advanced primary biliary cirrhosis at the University of Pittsburgh from March 1980 through September 1985. The transplant operation was relatively easy to perform. The most common technical complications experienced were fragmentation and intramural dissection of the recipient hepatic artery, which required an arterial graft in 20% of the cases. Most of the postoperative mortality occurred in the first 6 mo after transplantation, with an essentially flat actuarial life survival curve from that time point to a projected 5-yr survival of 66%. Common causes of death included rejection and primary graft nonfunction. Thirteen of the 76 patients had some hepatic dysfunction at the time of the last follow-up, although none were jaundiced. Recurrence of primary biliary cirrhosis could not be demonstrated in any of the patients. Antimitochondrial antibody was detected in the serum of almost all of the patients studied postoperatively for it. Most important, almost all of the 52 surviving patients have been rehabilitated socially and vocationally.