Timothy B. Plummer

Expression of tissue factor mRNA in cardiac xenografts: clues to the pathogenesis of acute vascular rejection.

BACKGROUND Acute vascular rejection destroys vascularized xenografts over a period of hours to days and is now considered the major hurdle to the clinical application of xenotransplantation. The hallmark of acute vascular rejection is diffuse intravascular coagulation; however, the pathogenesis of coagulation is a matter of controversy. One line of evidence points to activated endothelial cells and another to activated inflammatory cells as a source of tissue factor and thus as a primary cause of this lesion. The distinction between the two mechanisms inducing coagulation in the xenograft provides an opportunity for specific intervention. METHODS To explore these mechanisms, we studied the expression of tissue factor mRNA by in situ reverse transcriptase-polymerase chain reaction in relation to the histopathologic manifestations of acute vascular rejection in guinea pig hearts transplanted into rats treated by cobra venom factor to avoid the hyperacute rejection. RESULTS Three hours after transplantation and before the deposition of fibrin, tissue factor mRNA was expressed in the endothelial cells lining small and medium blood vessels and in smooth muscle cells of guinea pig cardiac xenografts. Sixteen hours after transplantation, while rat tissue factor mRNA was expressed only in occasional infiltrating cells, cardiac xenografts showed prominent deposits of fibrin in small vessels. Maximum expression of tissue factor on rat infiltrating cells was observed 48 hr after transplantation. CONCLUSIONS These results suggest that in acute vascular rejection, coagulation is initiated on the donor vascular system, while the procoagulant characteristics of infiltrating cells may reflect a response to tissue injury rather than a cause.

Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human‐pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens.

Infection-induced proteolysis of PGRP-LC controls the IMD activation and melanization cascades in Drosophila

The Drosophila immune deficiency (IMD) pathway, homologous to the mammalian tumor necrosis factor (TNF‐α) signaling pathway, initiates antimicrobial peptide (AMP) production in response to infection by gram‐negative bacteria. A membrane‐spanning peptidoglycan recognition protein, PGRP‐LC, functions as the receptor for the IMD pathway. This receptor is activated via pattern recognition and binding of monomeric peptidoglycan (DAP‐type PGN) through the PGRP ectodomain. In this article, we show that the receptor PGRP‐LC is down‐regulated in response to Salmonella/Escherichia coli infection but is not affected by Staphylococcus infection in vivo, and an ectodomain‐deleted PGRP‐LC lacking the PGRP domain is an active receptor. We show that the receptor PGRP‐LC regulates and integrates two host defense systems: the AMP production and melanization. A working model is proposed in which pathogen invasion and tissue damage may be monitored through the receptor integrity of PGRP‐LC after host and pathogen are engaged via pattern recognition. The irreversible cleavage or down‐regulation of PGRP‐LC may provide an additional cue for the host to distinguish pathogenic microbes from nonpathogenic ones and to subsequently activate multiple host defense systems in Dro‐sophila, thereby effectively combating bacterial infection and initiating tissue repair.—Schmidt, R. L., Trejo, T. R., Plummer, T. B., Platt, J. L., Tang, A. H. Infection‐induced proteolysis of PGRP‐LC controls the IMD activation and melanization cascades in Drosophila. FASEB J. 22, 918–929 (2008)

Apoptosis and Cellular Activation in the Pathogenesis of Acute Vascular Rejection

Abstract— Acute vascular or humoral rejection, a vexing outcome of organ transplantation, has been attributed by some to activation and by others to apoptosis of endothelial cells in the graft. We asked which of these processes causes acute vascular rejection by tracing the processes during the development of acute vascular rejection in porcine cardiac xenografts performed in baboons. Apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), expression of activated caspase-3, and proapoptotic genes Bax and Bcl-xL, was not detected until acute vascular rejection was well advanced, and even then, apoptosis was largely confined to myocytes. Activation of the endothelium, as evidenced by expansion of rough endoplasmic reticulum and increased ribosomal antigen and phospho-p70 S6 kinase, occurred early in the course of acute vascular rejection and progressed through the disease process. These findings suggest that acute vascular rejection is caused by an active metabolic process and not by apoptosis in the endothelium.

Immune Complex Formation after Xenotransplantation: Evidence of Type III as well as Type II Immune Reactions Provide Clues to Pathophysiology

Rejection of renal and cardiac xenografts is initiated when natural antibodies of the recipient bind to donor endothelium, activating complement on the surface of endothelial cells. Pulmonary xenotransplants, however, reveal less evidence of antibody binding and complement activation and, in contrast to other xenografts, fare worse when the complement of the graft recipient is depleted. Accordingly, we asked whether distinct immunochemical reactions might occur after xenotransplantation of the lung and what implications such reactions might have for pulmonary pathophysiology. Analysis of serum from baboons after transplantation with porcine lungs revealed complexes containing baboon IgM and porcine von Willebrand factor. The baboon IgM in these complexes was specific for Galalpha1-3Gal. Immune complexes were also seen, albeit to a lesser extent, in the serum of kidney and heart xenotransplant recipients. Deposits of porcine von Willebrand factor and baboon C3 were detected in livers and spleens of transplanted baboons. These results indicate pulmonary xenotransplantation eventuates in formation of immune complexes and in the deposition of those complexes at distant sites. Immune complex formation could explain the peculiar fate of xenoreactive antibodies after pulmonary xenotransplantation and might contribute to the pathophysiology of the lung and systemic changes not previously considered a complication of xenotransplantation.

An immunohistochemical survey for neuroendocrine cells in regional pancreatic lymph nodes: A plausible explanation for primary nodal gastrinomas?

BACKGROUND The existence of primary gastrinomas in lymph nodes continues to be controversial, because distinction from metastases from occult or regressed primary tumors may be difficult to exclude with certainty. If primary nodal gastrinomas do indeed occur, precursor neuroendocrine cells should be identifiable within lymph nodes. In a effort to detect these cells we undertook an immunohistochemical survey of regional pancreatic lymph nodes by using antibody against chromogranin, a highly specific marker for mature neuroendocrine cells. METHODS We retrospectively identified consecutive cases from five surgeons in which Whipple resections had been performed for nonendocrine pathologic conditions. All formalin-fixed, paraffin-embedded lymph nodes were identified from these 106 cases, excluding lymph nodes with metastases. Immunoperoxidase staining with monoclonal antibody against chromogranin A was performed on single tissue sections. All slides were reviewed by a single pathologist; chromogranin-positive cells were identified and, when possible, further evaluated immunohistochemically with additional neuroendocrine markers, including sequential synaptophysin staining. RESULTS A total of 1026 lymph nodes were available for review. Although well-formed intranodal chromogranin-positive nests were not identified, six nodes from three patients contained 13 strongly chromogranin-positive cells representing putative neuroendocrine cells. Mast cells showed weak nonspecific staining with chromogranin but were morphologically distinct. Perinodal adipose tissue contained a single paraganglion and several small neuroendocrine clusters. CONCLUSIONS Putative neuroendocrine cells are rarely (less than 1%) found in regional pancreatic lymph nodes. Although these may be embryonic rests and might represent precursors of primary nodal gastrinomas, specific hormones have yet to be identified in these cells. Although these data provide support for the concept of primary nodal gastrinomas, this remains a diagnosis of exclusion.

Intrinsic resistance of hepatocytes to complement-mediated injury

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.

In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing.

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400–600 copies of HPV 16, HeLa 229 cells with 10–50 copies of HPV 18, and SiHa cells with 1–2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.

Detection of hepatitis C virus RNA in formalin-fixed paraffin-embedded sections with digoxigenin-labeled cRNA probes.

Although recent studies have analyzed Hepatitis C (HCV) infections in liver tissue by in situ hybridization (ISH), many of these studies have been of limited diagnostic utility because of the low copy numbers of HCV in formalin-fixed paraffin-embedded (FFPE) tissue and failure to correlate the ISH analysis with other methods of detecting HCV.Thirty six cases of liver biopsies from patients with known HCV antibody status including 20 cases of serum HCV positive and 16 cases of serum HCV negative were analyzed. All cases showed histologic features suggestion of HCV infection. Analyses of all 36 cases were done by RT-PCR combined with Southern hybridization (RT-PCR-SH) and in situ hybridization (ISH). A prolactin riboprobe was used as a negative control. Immunohistochemistry (IHC) with an antibody against HCV (Rb 246) was also used to analyze HCV viral protein in the tissues. Of the 20 serum antibody-positive cases, RT-PCR-SH detected 18 positive cases, while ISH and IHC detected 19 and 16 positive cases, respectively. Of the 16 serum antibody-negative cases, RT-PCR-SH detected 8 positive cases while ISH and IHC detected 8 and 11 positive cases, respectively. A positive ISH signal for HCV was also detected in some lymphocytes and bile ducts in the liver. These results show that ISH with a highly specific riboprobe is comparable to RT-PCR-SH for detection of HCV infection in liver tissue.

Toward the survival and function of xenogeneic hepatocyte grafts

Xenogeneic hepatocyte transplantation might offer an unobtrusive alternative to whole liver allotransplantation. Having previously found that the immune response to such grafts can be controlled by immunosuppression, we sought approaches to collection and delivery that would optimize survival and function after transplantation. Porcine hepatocytes were isolated by a 2‐step collagenase technique and then: 1) used immediately; 2) stored in University of Wisconsin (UW) solution at 4°C; 3) cultured in supplemented Williams E medium; or 4) cryopreserved in UW solution with 10% dimethyl sulfoxide (DMSO). The fate and function of the hepatocytes was determined after they were injected into the spleens of immunodeficient mice. Freshly isolated hepatocytes had better viability (92.2 ± 1.9%) than hepatocytes cultured for 24 hours (78.4 ± 6.3%), hypothermically preserved in UW solution for 24 hours (85.8 ± 3.1%), or cryopreserved (65.0 ± 2.6%). Freshly isolated hepatocytes secreted more albumin after transplantation than hepatocytes that were cultured, hypothermically stored, or cryopreserved. In conclusion, culture and storage profoundly compromises the function of isolated hepatocytes after transplantation. Freshly isolated hepatocytes are the preferred source for transplantation. (Liver Transpl 2005;11:39–50.)