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-alpha) 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 Drosophila, thereby effectively combating bacterial infection and initiating tissue repair.

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.

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 degrees 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.

Acute vascular rejection and accommodation: divergent outcomes of the humoral response to organ transplantation.

BACKGROUND: The most difficult barrier to organ transplantation is humoral rejection, a condition initiated by binding of antibodies to blood vessels in the graft. Fortunately, humoral rejection is not the only outcome of antibody binding to the graft. In some cases, accommodation, a condition in which the graft does not undergo humoral injury despite the existence of humoral immunity directed against it, occurs and the graft remains seemingly inured. The mechanism underlying accommodation is uncertain, but changes in the function of antibodies, changes in the target antigen, and changes in the graft imparting resistance to injury have been implicated. METHODS: Using the swine-to-baboon cardiac xenograft model, we asked which mechanism(s) may distinguish acute vascular rejection from accommodation. RESULTS: In both acute vascular rejection and accommodation, antibodies were bound and complement activated in blood vessels of the graft. However, in acute vascular rejection, the full complement cascade was activated; while in accommodation, the complement cascade was interrupted, suggesting complement was inhibited in the latter condition. In acute vascular rejection, heparan sulfate and syndecan-4-phosphate, which can aid in complement control, were nearly absent, whereas in accommodation these were present in heightened amounts. CONCLUSION: These findings suggest that control of complement may underlie accommodation, at least in part, and raise the possibility that this control and possibly other protective mechanisms could be exerted by heparan sulfate.

Survival of Human Neurofibroma in Immunodeficient Mice and Initial Results of Therapy With Pirfenidone.

Neurofibromatosis type I is a common tumor predisposing disease in humans. Surgical therapy can be applied only in selected patients with resectable masses. Hence, development of new therapies for this disease is urgent. We used human neurofibroma implants in mice with severe combined immunodeficiency (SCID) as a model to test the toxicity and potential efficacy of pirfenidone, a new therapeutic agent. Two hundred twelve human neurofibromas were transplanted into various locations in 59 experimental animals, and 30 mice with implants received oral pirfenidone for up to six weeks. Survival of neurofibromas in animals treated with pirfenidone was lower than in the control group $(P=.02)$. Tumors did not change histologic appearance or vascularization in response to pirfenidone. Treatment with pirfenidone, a new antifibrotic agent, inhibits survival of some tumors without causing toxicity in animals.

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.

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. * differentiation * fusion * retrovirus

Apoptosis and cellular activation in the pathogenesis of acute vascular rejection.

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-x(L), 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.