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This paper describes the stages of osteogenic differentiation of chondrogenic graft in a culture medium and recipient bed (defect of bone tissue). In the culture medium, cell and matrix transformations, formation of vascular cavities with an endothelial lining and matrix vesicles in osteoblasts (the first stage of mineralization) take place in the chondrograft. The expression of chondrogenic genes and proteins is replaced by the expression of genes and proteins of the osteogenic stage of differentiation. Further stages of bone tissue histogenesis occur in the recipient bed. Humoral factors of regulation and ductile substance enter through the formed anastomosis of the vessels of osteodysplastica and recipient. On their basis, the formation of an organ-specific regenerate is completed with full integration into the recipient organism. These results are a starting point for further use of osteogenic grafts in clinical practice.

Unlike other carcinomas, hepatocellular carcinoma (HCC) metastasizes to distant organs relatively rarely. In contrast, it routinely metastasizes to liver vasculature/liver, affecting portal veins 3-10 times more often than hepatic veins. This portal metastatic predominance is traditionally rationalized within the model of a reverse portal flow, due to accompanying liver cirrhosis. However, this intuitive model is not coherent with facts: 1) reverse portal flow occurs in fewer than 10% of cirrhotic patients, while portal metastasis occurs in 30-100% of HCC cases, and 2) portal vein prevalence of HCC metastasis is also characteristic of HCC in non-cirrhotic livers. Therefore, we must assume that the route for HCC metastatic dissemination is the same as for other carcinomas: systemic dissemination via the draining vessel, i.e., via the hepatic vein. In this light, portal prevalence versus hepatic vein of HCC metastasis appears as a puzzling pattern, particularly in cases when portal HCC metastases have appeared as the sole manifestation of HCC. Considering that other GI carcinomas (colorectal, pancreatic, gastric and small bowel) invariably disseminate via portal vein, but very rarely form portal metastasis, portal prevalence of HCC metastasis appears as a paradox. However, nature does not contradict itself; it is rather our wrong assumptions that create paradoxes. The 'portal paradox' becomes a logical event within the hypothesis that the formation of the unique portal venous system preceded the appearance of liver in evolution of chordates. The analysis suggests that the appearance of the portal venous system, supplying hormones and growth factors of pancreatic family, which includes insulin, glucagon, somatostatin, and pancreatic polypeptide (HGFPF) to midgut diverticulum in the early evolution of chordates (in an Amphioxus-like ancestral animal), promoted differentiation of enterocytes into hepatocytes and their further evolution to the liver of vertebrates. These promotional-dependent interactions are conserved in the vertebrate lineage. I hypothesize that selective homing and proliferation of malignant hepatocytes (i.e., HCC cells) in the portal vein environment are due to a uniquely high concentration of HGFPF in portal blood. HGFPF are also necessary for liver function and renewal and are significantly extracted by hepatocytes from passing blood, creating a concentration gradient of HGFPF between the portal blood and hepatic vein outflow, making post-liver vasculature and remote organs less favorable spaces for HCC growth. It also suggested that the portal vein environment (i.e., HGFPF) promotes the differentiation of more aggressive HCC clones from already-seeded portal metastases, explaining the worse outcome of HCC with the portal metastatic pattern. The analysis also offers new hypothesis on the phylogenetic origin of the hepatic diverticulum of cephalochordates, with certain implications for the modeling of the chordate phylogeny.

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.

All carcinomas are characterized by metastases to distant organs, accounting for 90% of cancer-associated deaths. This analysis highlights a paradox of gastrointestinal carcinomas metastasis into portal vein: colorectal, pancreatic, small bowel and gastric carcinomas invariably release cancer cells to portal vein, which is the primary dissemination route, but very rarely form intraportal metastasis. On the contrary, hepatocellular carcinoma rarely releases cancer cells into portal vein, but very often forms intraportal metastasis. This disproportional frequency of HCC portal metastasis cannot be rationalized by hepatofugal (reverse) portal blood flow due to accompanying liver cirrhosis because: 1) reverse portal flow occurs in about 10% of cirrhotic patients, while portal metastasis occurs in 40-100% of HCC cases, and 2) portal vein prevalence of HCC metastasis is also the characteristic of non-cirrhotic patients. The ‘portal paradox’ becomes a logical event in light of comparative morphology and phylogeny of the chordate lineage: the formation of the unique portal venous system preceded the appearance of liver in evolution of chordates. The analysis suggests that the appearance of the portal venous system, draining blood from caudal intestine and supplying hormones and growth factors of pancreatic family (HGFPF) to midgut diverticulum in the early evolution of ancestral chordates, promoted differentiation of the diverticulum’s enterocytes into the hepatocyte phenotype and further formation of the liver in evolution of vertebrates. These promotional-dependent interactions are conserved in the vertebrate lineage but attributed to hepatocyte-derived cells only; the rest of gastrointestinal endodermal derivatives occurred independently from portal system in chordate phylogeny. Therefore, non-HCC gastrointestinal carcinomas’ cells do not have this affinity to and dependence on portal blood and portal blood does not promote attachment and growth of non-HCC gastrointestinal carcinomas’ cells in the portal conduit. I hypothesize that selective homing and proliferation of HCC cells in the portal vein environment are due to a uniquely high concentration of HGFPF in portal blood: portal vein formed by merging superior mesenteric and pancreatic veins. HGFPF are also necessary for liver function and renewal and are extracted by hepatocytes from passing blood, creating a concentration gradient of HGFPF between the portal blood and hepatic vein outflow, making post-liver organs less favorable spaces for HCC metastasis. This also suggests that the portal vein environment (i.e., high concentration of HGFPF) promotes the differentiation and selection of more aggressive HCC clones from already-seeded portal HCC cells, explaining the worse outcome of HCC patients with portal metastasis. Citation Format: Vladimir M. Subbotin. Overlooking the obvious - paradox of gastrointestinal carcinomas metastasis to portal vein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3460.

Clinical diagnosis is based on analysis of pathologic findings that may result in perceived patterns. The same is true for diagnostic pathology: Pattern analysis is a foundation of the histopathology-based diagnostic system and, in conjunction with clinical and laboratory findings, forms a basis for the classification of diseases. Any histopathology diagnosis is based on the explicit assumption that the same diseased condition should result in formation of the same (or highly similar) morphologic patterns in different individuals; it is a standard approach in microscopic pathology, including that of non-communicable chronic diseases with organ remodeling. During fifty years of examining diseased tissues under microscopy, I keep asking the same question: Why is a similarity of patterns expected for chronic organ remodeling? For infection diseases, xenobiotic toxicity and deficiencies forming an identical pathologic pattern in different individuals is understandable and logical: The same infection, xenobiotic, or deficiency strikes the same target, which results in identical pathology. The same is true for Mendelian diseases: The same mutations lead to the same altered gene expressions and the same pathologic pattern. But why does this regularity hold true for chronic diseases with organ remodeling? Presumable causes (or risk factors) for a particular chronic disease differ in magnitude and duration between individuals, which should result in various series of transformations. Yet, mysteriously enough, pathological remodeling in a particular chronic disease always falls into a main dominating pattern, perpetuating and progressing in a similar fashion in different patients. Furthermore, some chronic diseases of different etiologies and dissimilar causes/risk factors manifest as identical or highly similar patterns of pathologic remodeling. HYPOTHESIS: I hypothesize that regulations governing a particular organ's chronic remodeling were selected in evolution as the safest response to various insults and physiologic stress conditions. This hypothesis implies that regulations directing diseased chronic remodeling always preexist but normally are controlled; this control can be disrupted by a diverse range of non-specific signals, liberating the pathway for identical pathologic remodeling. This hypothesis was tested in an analysis of arterial neointimal formation, the identical pathology occurring in different diseases and pathological conditions: graft vascular disease in organ transplantation, in-stent restenosis, peripheral arterial diseases, idiopathic intimal hyperplasia, Kawasaki disease, coronary atherosclerosis and as reaction to drugs. The hypothesis suggests that arterial intimal cells are poised between only two alternative pathways: the pathway with controlled intimal cell proliferation or the pathway where such control is disrupted, ultimately leading to the progressive neointimal pathology. By this property the arterial neointimal formation constitutes a special case of Kauffman's self-organization. This new hypothesis gives a parsimonious explanation for identical pathological patterns of arterial remodeling (neointimal formation), which occurs in diseases of different etiologies and due to dissimilar causes/risk factors, or without any etiology and causes/risk factors at all. This new hypothesis also suggests that regulation facilitating intimal cell proliferation cannot be overwritten or annulled because this feature is vital for arterial differentiation, cell renewal, and integrity. This hypothesis suggests that studying numerous, and likely interchangeable, non-specific signals that disrupt regulation controlling intimal cell proliferation is unproductive; instead, a study of the controlling regulation(s) itself should be a priority of our research.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.

Hepatocellular carcinoma (HCC) disseminates systemically, but metastases occur in distant organs only in minority of patients, whereas HCC routinely metastasizes to liver and its vessels. HCC cells disseminate via hepatic veins, but portal veins are affected by metastasis more frequently than are hepatic veins, and correlates with poor prognosis. In this review, I suggest that privileged HCC portal metastasis occurs because of high levels of pancreatic family hormones and growth factors (PHGFs) in the portal blood. The analysis suggests that the appearance of the portal system carrying PHGFs in the evolution of invertebrate chordate (Amphioxus) led to the evolution of the liver in vertebrate; given that the portal pattern of HCC metastasis and selection of more-aggressive clones are PHGF dependent, PHGFs and their ligands constitute therapeutic targets.

Adolescent idiopathic scoliosis (AIS) affects 2–3% of children. Numerous hypotheses on etiologic/causal factors of AIS were investigated, but all failed to identify therapeutic targets and hence failed to offer a cure. Therefore, currently there are only two options to minimize morbidity of the patients suffering AIS: bracing and spinal surgery. From the beginning of 1960th, spinal surgery, both fusion and rod placement, became the standard of management for progressive adolescent idiopathic spine deformity. However, spinal surgery is often associated with complications. These circumstances motivate AIS scientific community to continue the search for new etiologic and causal factors of AIS. While the role of the genetic factors in AIS pathogenesis was investigated intensively and universally recognized, these studies failed to nominate mutation of a particular gene or genes combination responsible for AIS development. More recently epigenetic factors were suggested to play causal role in AIS pathogenesis. Sharing this new approach, we investigated scoliotic vertebral growth plates removed during vertebral fusion (anterior surgery) for AIS correction. In recent publications we showed that cells from the convex side of human scoliotic deformities undergo normal chondrogenic/osteogenic differentiation, while cells from the concave side acquire a neuronal phenotype. Based on these facts we hypothesized that altered neural crest cell migration in early embryogenesis can be the etiological factor of AIS. In particular, we suggested that neural crest cells failed to migrate through the anterior half of somites and became deposited in sclerotome, which in turn produced chondrogenic/osteogenic-insufficient vertebral growth plates. To test this hypothesis we conducted experiments on chicken embryos with arrest neural crest cell migration by inhibiting expression of Paired-box 3 (Pax3) gene, a known enhancer and promoter of neural crest cells migration and differentiation. The results showed that chicken embryos treated with Pax3 siRNA (microinjection into the neural tube, 44 h post-fertilization) progressively developed scoliotic deformity during maturation. Therefore, this analysis suggests that although adolescent idiopathic scoliosis manifests in children around puberty, the real onset of the disease is of epigenetic nature and takes place in early embryogenesis and involves altered neural crest cells migration. If these results confirmed and further elaborated, the hypothesis may shed new light on the etiology and pathogenesis of AIS.

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190–230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%–90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model. Mol Cancer Ther; 17(1); 140–9. ©2017 AACR.

Insufficient drug uptake by solid tumors remains the major problem for systemic chemotherapy. Many studies have demonstrated anticancer drug effects to be dose-dependent, although dose-escalation studies have resulted in limited survival benefit with increased systemic toxicities. One solution to this has been the idea of loco-regional drug treatments, which offer dramatically higher drug concentrations in tumor tissues while minimizing systemic toxicity. Although loco-regional delivery has been most prominent in cancers of the liver, soft tissues and serosal peritoneal malignancies, survival benefits are very far from desirable. This review discusses the evolution of loco-regional treatments, the present approaches and offers rapidly reversible hydrophobization of drugs as the new future direction.

Anticancer dendritic cells (DC) therapy currently uses in vitro propagation of the patient's DC and pulsing with tumor antigens. However, clinical achievements are far from desirable. Here, I suggest that the lack of anticipated responses could be because cancer cells continuously mutate, whereas the population of tumor antigens from the excised tumor is genetically static, and because there is an absence of biologic mechanisms to facilitate intratumoral DC retention, which is needed for DC pulsing. I hypothesize that stable tumor transfection with fetal liver tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) DNAs will induce homing, propagation and maturation of intratumoral DC. This must be followed by drug-induced apoptosis of tumor cells, to ensure the release of tumor antigens for DC pulsing. Then, regardless of any mutation of tumor cells, they would always incite DC propagation and maturation, pulsing and antitumor immunity.

The consensus hypothesis on coronary atherosclerosis suggests high LDL-C levels as the major cause and pursues it as the therapeutic target, explicitly assuming: (i) tunica intima of human coronaries consists of only one cell layer – endothelium, situated on a thin layer of scarcely cellular matrix; and (ii) subendothelial lipoprotein retention initiates the disease. Facts showed: (i) normal tunica intima invariably consists of multiple cellular layers; and (ii) initial lipid depositions occurred in the deepest layers of tunica intima. This review suggests that coronary atherosclerosis starts with pathological intimal expansion, resulting in intimal hypoxia and neovascularization from adventitial vasa vasorum, facilitating lipoprotein extraction by previously avascular deep intimal tissues. Until the hypothesis incorporates real knowledge, our efforts will probably be off-target.

Previously, we established a model in which physiologically adequate function of the autologous β cells was recovered in non-obese diabetic (NOD) mice after the onset of hyperglycemia by rendering them hemopoietic chimera. These mice were termed antea-diabetic. In the current study, we addressed the role of T regulatory (Treg) cells in the mechanisms mediating the restoration of euglycemia in the antea-diabetic NOD model. The data generated in this study demonstrated that the numbers of Treg cells were decreased in unmanipulated NOD mice, with the most profound deficiency detected in the pancreatic lymph nodes (PLNs). The impaired retention of the Treg cells in the PLNs correlated with the locally compromised profile of the chemokines involved in their trafficking, with the most prominent decrease observed in SDF-1. The amelioration of autoimmunity and restoration of euglycemia observed in the antea-diabetic mice was associated with restoration of the Treg cell population in the PLNs. These data indicate that the function of the SDF-1/CXCR4 axis and the retention of Treg cells in the PLNs have a potential role in diabetogenesis and in the amelioration of autoimmunity and β cell regeneration in the antea-diabetic model. We have demonstrated in the antea-diabetic mouse model that lifelong recovery of the β cells has a strong correlation with normalization of the Treg cell population in the PLNs. This finding offers new opportunities for testing the immunomodulatory regimens that promote accumulation of Treg cells in the PLNs as a therapeutic approach for type 1 diabetes (T1D).

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Hepatic levels of a powerful vasoconstrictor endothelin-1 (ET-1) and its receptors increase in human and carbon tetrachloride (CCl4)-induced liver cirrhosis. The aim of this study was to determine whether antagonism of hepatic ET-1 receptors ameliorates CCl4-induced hepatic injury and portal hypertension in rats. Acute liver injury was induced by a single intraperitoneal injection of CCl4 (0.3 ml/kg), whereas cirrhosis and portal hypertension were induced by CCl4 treatment (0.15 ml/kg twice a week) for 8 weeks. Hepatic morphology, ET-1 and its receptors, and portal venous pressures were determined. Increases in ET-1 and its receptors occurred within 24 h of CCl4 administration, and progressively thereafter during the development of cirrhosis. The acute CCl4-induced hepatic injury was characterized by significant increases in portal pressure (from 8.7+/-1.8 to 17.6+/-3.3 mmHg; p

Natural IgM antibodies (Abs) play an important role in clearing pathogens, enhancing immune responses, and preventing autoimmunity. However, the molecular mechanisms that mediate the functions of natural IgM Abs are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs are commonly polyspecific and recognize a variety of antigens (Ags) with no apparent structural homology. It is generally believed that polyspecificity is an inherent property of natural Abs. However, there is increasing evidence that polyspecificity may be induced by mild denaturing conditions. In this study, we compared the specificity of three polyspecific IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All three Abs lost their polyspecificity in serum. They no longer reacted with conventional screening Ags, including hapten-BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with cognate peptide Ags selected from a T7 phage library. The acquisition of narrow specificity by polyspecific IgM in serum was also observed with muscle tissue sections used as a source of endogenous Ags. The loss of polyspecificity by different Abs was apparently dependent on the presence of different serum constituents. The results of this study suggest that the seemingly inherent polyspecificity of many natural IgM Abs may be largely an in vitro phenomenon related to the lack of normal serum components in the medium. Potential mechanisms underlying the loss of polyreactivity are discussed.

SummaryWe have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.

We have recently developed a simple and highly efficient methodology for delivering plasmid DNA (pDNA) to skeletal muscle cells of mammalian limbs. The procedure involves the rapid intravascular injection of a large volume of saline (containing pDNA) into the vasculature of the distal limb. As a result of the robust delivery methodology involved, it is important to understand the effects of the injection procedure on the skeletal muscle tissue in the targeted limb. In previous studies, only modest and transient muscle damage was noted. In this study we quantitatively assessed the degree of muscle damage in rat limbs following intravascular injections using muscle histology (HE staining), membrane integrity (Evans blue staining), and leukocyte infiltration (immunohistochemistry) assays. The rapid extravasation of fluid during the injection process resulted in edema of the muscle tissue of the targeted limb; however, the edema was transient and resolved within 24 h. Consistent with observations from previous studies, minimal levels of myofiber damage were detected. Immunohistochemical labeling indicated that increased numbers of neutrophils (CD43+) and macrophages (ED1+ and ED2+) were present in the muscle tissue interstitium shortly after injection but that elevations were relatively modest and resolved by 2 weeks postinjection.

Background The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA. Methods We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance. Results Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20–60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them. Conclusions Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression. Copyright © 2006 John Wiley & Sons, Ltd.

Background and aims: Hepatic concentrations of the powerful vasoconstrictor and fibrogen endothelin 1 (ET-1) and its receptors increase in human and experimental cirrhosis, suggesting a major role for ET-1 in the pathology of chronic liver disease. We investigated whether ET-1 receptor antagonism, after the development of fibrosis and cirrhosis, arrests/reverses the progression of chronic liver disease. Methods: Chronic liver injury was induced in rats by carbon tetrachloride (CCl4) treatment (0.15 ml/kg intraperitoneally twice a week) in conjunction with phenobarbital in drinking water (0.4 g/l) for four (group 1: fibrosis) and eight (group 2: cirrhosis) weeks. Rat were then treated concurrently with the ET-1 receptor antagonist TAK-044 (10 mg/kg/day) and CCl4/phenobarbital for a further four weeks. Results: Histopathological examination revealed significant arrest of progression to cirrhosis in group 1 and reversal of cirrhosis in group 2 rats. TAK-044 treatment caused significant amelioration of portal hypertension, systemic hypotension, and liver injury (reduced activities of serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), and improved hepatic synthetic capacity (increased serum albumin concentration) in both groups of rats relative to vehicle treated rats. TAK-044 treatment reduced collagen synthesis, as evidenced by decreased hepatic hydroxyproline content, mRNA expression of collagen-α type I, and tissue inhibitors of matrix metalloproteinases 1 and 2, and mRNA and protein expression of a potent fibrogenic cytokine, transforming growth factor β1. Conclusions: The results emphasise the role of ET-1 in the development of cirrhosis and strongly suggest that blockade of its actions can be a rational therapy for chronic liver disease and its complications.

The liver is a major site for the synthesis and actions of platelet activating factor (PAF), a potent hepatic vasoconstrictor and systemic vasodilator. As PAF is implicated in portal hypertension and hyperdynamic circulation associated with liver cirrhosis, we characterised changes in the hepatic PAF system in experimental cirrhosis.In rats made cirrhotic by carbon tetrachloride (CCl(4)) administration for eight weeks, we determined hepatic levels of PAF and its cognate receptor, and the effects of PAF and PAF antagonist (BN52021) on portal and arterial pressure.Compared with control rats, cirrhotic rats had higher hepatic PAF levels, higher apparent hepatic efflux of PAF, and higher PAF levels in arterial blood (p0.01, p0.01, p0.05, respectively). Relative to controls, cirrhotic livers had elevated hepatic PAF receptors (by mRNA and protein levels and [(3)H]PAF binding), higher (p0.01) baseline hepatic portal pressure, and an augmented (p = 0.03) portal pressure response to PAF infusion (1 microg/kg). Portal infusion of BN52021 (5 mg/kg) showed that elevated endogenous PAF was responsible for 23% of the cirrhotic portal pressure increase but made no contribution to systemic hypotension. Finally, increased PAF receptor density was observed in the contractile perisinusoidal stellate cells isolated from cirrhotic livers relative to those from control livers.In cirrhosis, increased hepatic release of PAF elevates systemic PAF; in combination with upregulated hepatic PAF receptors in stellate cells, this contributes to portal hypertension.

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

Introduction: Given its multifactorial etiology, the relative contribution of anti-donor cellular and humoral immune responses in the pathogenesis of chronic rejection is as yet ambiguous. We hypothesized that alloreactive T and B cells play a seminal role in the development of this lesion. Methods: To address this hypothesis, RAG-2 −/− mice were used as donors and recipients in a well-established murine model of aortic transplantation. Grafts were transplanted across the following groups: Group I: C3H → C3H; Group II: Wild-type [WT] 129Sv (H-2 b ) → C3H (H-2 k ); Group III: C3H → WT 129Sv; Group IV: 129SvEv RAG-2 −/− → C3H; and Group V: C3H → 129SvEv RAG-2 −/− . Grafts were harvested at d40 to 146 post-transplantation for morphologic and immunohistochemical analyses and semi-quantitative RT-PCR was employed to evaluate the intragraft mRNA expression of various immune mediators. Mixed lymphocyte reaction and complement-mediated alloantibody cytotoxicity assays were performed to determine anti-donor proliferative and humoral responses, respectively. Results: Unlike that across the syngeneic combination (Group I), marked intimal thickening with corresponding luminal narrowing was observed in the majority of the aortic allografts (Groups II–IV). On the contrary, the morphology of C3H aortic allografts harvested from the majority of the RAG-2 −/− was remarkably preserved. Correspondingly, anti-donor proliferative and humoral immune responses were undetectable in C3H → RAG-2 −/− recipients as was the intragraft mRNA expression of the Th 1 and the Th 2 -type cytokines. Conclusions: Taken together, these data suggest that in this murine model of aortic allotransplantation, donor-specific cellular and humoral responses play a dominant role in the initiation and perpetuation of chronic rejection.

Bone marrow transplantation (BMT) may induce tolerance across xenogeneic barriers. We have established a xenogeneic BMT model where hamster BM is transplanted into splenectomized LEW rat recipients resulting in high levels of engraftment. Unfortunately, graft vs. host disease (GVHD) with severe dermatitis developed in all rat recipients. We were successful in treating or preventing the dermatitis of this xenogeneic GVHD by the use of the T-cell suppressant tacrolimus. However, this compound did not prevent the development of a fatal liver injury in the rat recipients. This study was designed to elucidate the pathogenesis of this liver injury appearing in T-cell suppressed rat recipients of hamster BM. Splenectomized and irradiated (10 Gy) LEW rats received 300 × 106 unfractionated hamster BM cells. These BMT recipients were divided in 3 groups: Group I recipients (n = 8) did not receive further immunosuppression. Group II animals (n = 10) received tacrolimus 1 mg/kg/d for 7 d. Group III recipients (n = 6) were given the same daily dose of tacrolimus on a long-term basis. Chimerism was detected by flow cytometry. Cytotoxicity of recipient's sera against rat and hamster lymph node cells was measured by complement-dependent cytotoxicity (CDC) test. Immunofluorescence was used to detect hamster antirat antibodies on several recipient organs. In Group I, 2 out of 8 animals engrafted (25%) and survived for a median of 21 d showing the severe dermatitis characteristic of GVHD. In group II (n = 10), 9/10 rat recipients engrafted (90%) and survival was increased to a median of 53.7 days. However, these surviving recipients developed fatal GVHD not different from that observed in Group I recipients. All animals in Group III (n = 6) engrafted and did not show the characteristic dermatitis of GVHD. Their survival, however, was shortened to a median of 30.3 d by a severe liver injury. This injury was characterized by hepatocyte necrosis in zones 1 and 2 with polymorphonuclear (PMN) cell infiltration. Deposits of hamster immunoglobulins were present around the necrotic areas and in the portal veins. Moreover, antirat antibodies appeared in the circulation. These antibodies were sensitive to dithiothreitol (DTT) treatment indicating that they were of the IgM class. This study shows that xenogeneic GVHD may have a dual presentation in the hamster-to-rat model: a classical cellular GVHD not distinct to the allogeneic one and a humoral GVHD affecting solely the recipient liver. The degree of humoral injury is potentiated by T-cell suppression.

We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.

Background High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter-driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression. Methods Histological methods were used to determine the presence and extent of liver damage and hepatocyte proliferation. Viral and liver-specific promoters were tested to study promoter shut-off, Southern blotting was performed to determine the loss of the pDNA vector over time, and several mouse models were used to study the host immunological response. Results pDNA is lost rapidly early after injection, but remains at a relatively stable copy number after Day 4. Southern blotting experiments showed that plasmid DNA could be detected for at least 12 weeks after injection (0.2 copies per genome). The early rapid decline of expression is promoter dependent. A liver-specific albumin promoter resulted in similar levels of expression on Days 1 and 7, suggesting that promoter inactivation may be responsible for the instability of CMV promoter-driven expression. The slow decline in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much prolonged in immunosuppressed, immunodeficient, or antigen-tolerized mice. Conclusion The present data suggest that if promoter inactivation can be overcome, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo. Copyright © 2001 John Wiley & Sons, Ltd.

Infectious complications are the most frequent and severe complications of acute narcotizing pancreatitis (AP) with a mortality rate up to 80%. Although experimental and clinical studies suggest that the microbiologic source of pancreatic infection could be enteric, information in this regard is scant. This study evaluated bacterial translocation (BT) using mild and severe models of AP. Mild AP was induced by 6-hr continuous intravenous infusion of cerulein, while severe AP was induced by additional infusion of glycodeoxycholic acid into the biliopancreatic duct. BT was evaluated with organ cultures performed when animals were killed (24 hr). To confirm the gastrointestinal origin of the translocating microorganisms, fluorescent microspheres were also given to the animals in drinking water 24 hr before induction of AP. At the time of death beads were counted with a (fluorescence-activated cell sorter) (FACS) in peritoneal lavages and with fluorescent microscopy in frozen sections of the pancreata. Morphology of the distal small bowel showed significant changes in the animals with AP compared to controls, such as reduction of villus high and altered microvasculature. Mild AP induced BT to the pancreas in 100% of the animals, compared to pancreata from control groups. Severe AP induced increased BT to the pancreas. BT to liver and spleen was also significantly increased with AP. The presence of fluorescent microspheres confirmed their enteric derivation. This study provides evidence for the enteric origin of microorganisms responsible for pancreatic infectious complications during AP. The evidence of BT after laparotomy suggests an increased risk of infections with the association of these conditions. This could provide an explanation for the high mortality associated with laparotomy in course of AP.

Background/Aims: Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis. Methods: The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats. Results: ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered. Conclusions: These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.

Interleukin-12 (IL-12) and Flt3 ligand (FL) regulate hematopoiesis by stimulating proliferation, differentiation and migration of progenitor and/or stem cells. In this study, we evaluated the in vivo effect of IL-12 alone or in combination with FL on dendritic cell (DC) generation and accumulation in murine spleen, lymph node, thymus, liver, and tumor tissues. Both cytokines induced accumulation of functional DC in lymphoid and non-lymphoid tissues. IL-12 promoted predominantly myeloid dendropoiesis, while FL induced both myeloid and lymphoid dendropoiesis. Combination treatment resulted in a dramatic increase in CD86+, and particularly, NLDC-145+ cells within the liver, which was largely due to cell proliferation. Combination therapy also revealed the ability of FL to protect bone marrow cell populations from IL-12-induced depletion in vivo. In vitro, we found a significant FL-induced up-regulation of IL-12 production by DC at both mRNA and protein levels. Thus, our study suggests that (i) the antitumor activity of IL-12 may, at least in part, be mediated by the stimulation of dendropoiesis and (ii) IL-12 might contribute to the antitumor activity of FL. Furthermore, induction of DC generation in vivo by a combination of IL-12 and FL might become a new approach for immunotherapy of cancer.

Aim: The hypothesis that interleukin-6-IL-6/gp130 signaling is involved in liver and biliary epithelial cell (BEC) biology and growth control was tested by subjecting homozygous IL-6 deficient mice (IL-6 -/ -) and wild type (IL-6 +/+ ) littermate controls to bile duct ligation (BDL). Materials and methods: During the first week after BDL, the two groups were compared with respect to routine liver injury tests, liver histology, BEC and hepatocyte DNA synthesis, together with the expression of mRNA and protein of IL-6 as well as related growth factors, and their receptors. Results: During the first week after BDL, there was marked upregulation of IL-6 mRNA and protein in the IL-6 +/+ mice only in the vicinity of the biliary tree; whereas, biliary/peri-biliary IL-6R, HGF and met mRNA and protein increased in both groups. IL-6, HGF mRNA and protein localized to periductal inflammatory cells and stellate cells, while met and IL-6R protein were upregulated in the BEC and, to a lesser extent, in hepatocytes. This occurred during maximal proliferation of the BEC. Despite the absence of IL-6 in the IL-6 -/- mice, there were only mildly phenotypic differences between the two groups, and no differences in mortality. Compared to IL-6 +/+ controls, IL-6 -/- mice showed slightly less BEC proliferation, a trend toward more liver injury, and significantly higher total serum bilirubin (TB) levels, suggestive of impaired biliary tree integrity. These changes were associated with slightly less HGF mRNA and protein expression in the IL-6 -/- mice, but the differences were not significant. Leukemia inhibitory factor (LIF), another gp-130 ligand, also showed marked peri-biliary upregulation after BDL in both groups, and also induced BEC DNA synthesis, in vitro. Conclusions: The mild phenotypical differences between IL-6 +/+ and IL-6-'- mice in the acute response to BDL is most likely attributable to the redundancy of the gp-130 signaling system. However, the long-term response to BDL results in a distinct phenotype in the IL-6 -/- mice, marked by a relentless rise in serum total bilirubin and an inability to maintain compensatory increase in liver mass.

Following the initial demonstration that intramuscularly-injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor-mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented.

An abrogation of the decline in epidermal Langerhans cell numbers above melanoma might significantly improve the efficacy of immunotherapy for melanoma treatment. Systemic Flt3 ligand (FL) administration in mice induced a significant increase in mature dendritic cells (DC) within the skin, preferentially in the dermis, whereas IL-12 promoted a significant increase of immature DC preferentially in the epidermis. Both effects were abrogated in IL-12 knockout mice. Thus, IL-12 could promote FL-induced accumulation of skin DC. The involvement of FL and IL-12 in the regulation of DC accumulation within the skin may contribute, at least in part, to the stimulation of antimelanoma immunity by FL- and IL-12-based immunotherapies. Moreover, FL and IL-12 could be used for selective in vivo generation of DC in either epidermis or dermis for experimental and clinical purposes.

Pyruvate has been shown to prevent intestinal mucosal injury after ischemia-reperfusion. The aim of the present study was to determine whether pyruvate can (1) prevent postreperfusion mucosal injury occurring after intestinal preservation and subsequent transplantation and (2) exert a protective effect on the intestinal graft mucosa during acute rejection. Preservation mucosal injury was evaluated, after 2 hours of reperfusion, by comparing grafts transplanted in a rat syngeneic combination (ACI to ACI) after 2 hours of cold preservation using pyruvate (n = 6) or placebo (n = 6). Mucosal parameters obtained during acute rejection (allogeneic combination: ACI to Lewis) were compared between placebo-treated (n = 6) and pyruvate-treated (nequals 6) animals. Tissue injury was evaluated by histopathologic examination, oxygen free radical production by luminol-enhanced chemiluminescence, and degree of neutrophil infiltration by myeloperoxidase staining. After reperfusion of the preserved grafts and during acute rejection, mucosal oxygen free radical levels and the number of infiltrating neutrophils were significantly (P0.05) increased in the untreated grafts, whereas there was a statistically significant inhibition of these parameters in those treated with pyruvate. Mucosal injury, seen after reperfusion of the preserved grafts, was prevented by pyruvate. The histopathologic abnormalities observed in the untreated grafts during rejection were also significantly reduced by pyruvate. Treatment with pyruvate before cold preservation of intestinal grafts, in this rat model, reduced reperfusion mucosal injury, neutrophil infiltration, and oxygen free radical production. Oxygen free radicals were produced in the mucosa of the graft during acute rejection and their production was reduced by pyruvate, which exerted a protective effect on the rejecting allograft mucosa.

Guinea-pig livers are poorly reperfused when transplanted into rats. We have observed that, in contrast to that of the rat, the guinea-pig intrahepatic portal vein (PV) has a thick layer of smooth muscle. It is possible that, after perfusion of the liver with ice-cold saline, this could go into spasm, resulting in poor reperfusion. To test this hypothesis, guinea-pig livers were perfused with different solutions stored at varying temperatures and transplanted into LEW rats. To prevent xenograft hyperacute rejection, all xenograft recipients were treated with 80 U/kg cobra venom factor (CVF) i.v. on days -1 and 0. In addition to the percentage reperfusion, PV resistance and recipient survival were also monitored. In group I, liver xenografts perfused with ice-cold saline (4 degrees C) reperfused poorly (20-30%), resulting in the development of portal hypertension (16.5 cmH2O vs. 12 cmH2O in naive LEW rats) and shortened mean survival time (11.7 +/- 4.2 h). In contrast, group II livers perfused with saline at room temperature (23 degrees C) underwent homogeneous reperfusion (98-100%) with no increase in portal vein resistance, indicating that low temperature was the main trigger for the spasm of the PV. Moreover, recipient survival in this group was significantly prolonged to a mean of 22 + 2.6 h (P < 0.01). Although UW solution (group III) and the vasodilator sodium nitroprusside (NP) (group IV) when used alone improved the degree of hepatic reperfusion, it was still not optimal. The supplementation, however, of UW solution with NP in group V animals resulted in homogeneous reperfusion (98%) with no portal hypertension and consistent prolonged graft survival of 21.0 +/- 1.7 h. Therefore, this study has determined that the riddle of the abnormal reperfusion of guinea-pig liver xenografts by rat blood is nonimmune mediated and is due to the spasm of the strong smooth muscle in the PV tree produced by cold perfusates.

FLT3-ligand (FL) is a recently described cytokine that stimulates the proliferation and differentiation of hematopoietic progenitors both in vivo and in vitro and, when administered to mice, induces an accumulation of dendritic cells (DC) in different lymphoid and nonlymphoid organs and tissues, including the liver. We have studied the antitumor effect of FL administered alone or in combination with IL-12 in a day 3 murine liver metastasis model. FL significantly reduced the number of hepatic metastases (36.00 ± 11.00 vs 92.00 ± 10.19 in control group, p < 0.05). Histologic evaluation of the livers revealed that FL induced a significant infiltration of the tumor border by lymphocytes and DC associated with increased number of apoptotic figures. Immunohistochemical analysis demonstrated that FL significantly enhanced the number of DC in the liver parenchyma and within the liver metastases, as well as the number of CD4+ and CD8+ T lymphocytes. These data support the suggestion that DC may be directly involved in the antitumor effect of FL. Interestingly, the antitumor effect of FL was greatly reduced by the NK depletion. Combination of FL and IL-12 resulted in greater antitumor efficacy than these cytokines alone. In summary, we have shown that FL has significant antitumor effect on preexisting murine C3 liver tumors that is mediated by NK cells. We have also demonstrated that the FL/IL-12 combination has an enhanced antitumor activity in the same murine tumor model.

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of noncytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. infusion of 50 x 10(6) donor BM cells. They were given FL (10 microg/day i.p., x7 days), tacrolimus (2mg/kg/day i.p., x13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days). The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cells against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus +/- FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly increased in conventionally immunosuppressed organ allograft recipients given donor BM + a potent haematopoietic and DC-growth promoting cytokine. Although withdrawal of systemic immunosuppression appears to allow exhibition of the potential allostimulatory activity of these donor APC leading to rejection, the model provides a useful basis for further evaluation of the persistence and manipulation of donor haematopoietic cells and in particular, donor-derived APC, on the outcome of organ transplantation.

Aortic allotransplantation is a reliable procedure to study the evolvement of chronic rejection in mice. The progressive nature of this process in mice is characterized by diffuse and concentric myointimal proliferation which is inevitably associated with variable degrees of luminal constriction. These vascular changes are comparable to those that are witnessed in organ allografts undergoing chronic rejection in humans, underscoring its utility as a model of choice for the study of the development of this lesion. Whilst improved surgical technique has resulted in markedly enhanced graft survival, the results are far from being acceptable. Realizing this limitation, we embarked on developing a modified technique for aortic transplantation which would allow for improved graft survival in mice. A bypass conduit was created by end-to-side anastomosis of a segment of the donor's thoracic aorta into the infrarenal portion of the recipient's abdominal aorta. Using this technique, the graft survival was >98% with evidence in allotransplanted aorta of morphological changes pathognomonic of chronic rejection. On the contrary, no histopathological anomalies were discerned in aortic grafts transplanted across syngeneic animals. This modified surgical approach ameliorates the unacceptably high graft loss associated with earlier techniques, further extending the utility of this model as a tool to study the molecular and cellular mechanisms rudiment to the evolvement of chronic rejection.

Background In this study, using a murine model of aortic allotransplantation, the role of blockade of signaling through CD28/B7 and CD40/CD40 ligand co-stimulatory pathways in the evolvement of posttransplant vasculopathy was examined. Methods. Aortic allografts were transplanted across C57BL/10J (H2 b )→C3H (H2 k ) strain combinations. Transient or more stable blockade of second signaling was achieved by either a single injection or multiple injections of CTLA4-Ig fusion protein (200 μg/dose i.p.) and/or anti-CD40 ligand (CD40L) monoclonal antibody (250 μg i.m.). At day 30 after transplantation, the grafts were harvested for histopathological and immunohistochemical examination. Results. Similar to allografts of untreated animals, aortic allografts obtained from recipients treated with either CTLA4-Ig or anti-CD40L monoclonal antibody alone exhibited marked narrowing of the lumen primarily due to concentric intimal thickening caused by proliferation of α-smooth muscle actin-positive cells. Contemporaneous treatment, however, with either a single injection or multiple injections of CTLA4-Ig and anti-CD40L monoclonal antibody resulted in marked diminution of intimal thickening. Interestingly, concurrent prolonged inhibition of CD28/B7 and CD401 CD40L pathways resulted in complete abrogation of the development of posttransplant arteriopathy. Conclusion. These data suggest that a more stable disruption of signaling through costimulatory pathways may be required to obviate the development of posttransplant vasculopathy.

Aortic allotransplantation in mice has been well established as a model of choice to study the evolvement of chronic rejection, the etiopathology of which is believed to be that of immune origin. This has prompted the postulation that prior induction of donor-specific tolerance would attenuate or abrogate the underlying events that culminate in posttransplant arteriosclerosis. To study the effects of donor-specific tolerance on chronic rejection, we performed orthotopic liver transplantation without immunosuppression in mice 30 days before aortic allotransplantation across C57Bl/ 10J (H2b)-->C3H (H2k) strain combinations (group III). Aortic allografting in syngeneic (group I; C3H-->C3H) and allogeneic (group II, C57Bl/10J-->C3H) animals served as controls. No morphological changes were evidenced in the transplanted aortas in group I animals. Contrarily, aortic allografts in group II animals underwent a self-limiting acute cellular rejection, which resolved completely and was succeeded by day 30 after transplantation by histopathological changes pathognomonic of chronic rejection. There was evidence for diffuse myointimal thickening, progressive concentric luminal narrowing, and patchy destruction of internal elastic membranes resulting in massive vascular obliteration by day 120 after transplantation. It was of interest that no arteriosclerotic changes were observed for the duration of follow-up (up to 120 days after transplantation) in transplanted aortas (liver donor-type) harvested from animals in group III. However, vasculopathy was prominent in third-party aortic grafts transplanted into tolerant recipients. Taken together, these data suggest that prior induction of tolerance abrogates the development of chronic rejection; this protection seems to be donor specific.

In organ allograft recipients, the persistence of donor leukocytes within recipient tissues for indefinite periods has been construed as evidence of the transfer of hematopoietic stem and progenitor cells (1, 2). Moreover, multilineage donor leukocyte microchimerism has been postulated to be an essential condition for organ allograft acceptance (1–3). Accounts of the multilineage nature of microchimerism have emphasized the invariable prominence of donor-derived dendritic cells (DC*) (1–5). Both DC progenitors and other leukocytes of donor origin that engage the host immune system may have tolerogenic properties (6). Current efforts to enhance “spontaneous microchimerism” in human organ transplantation focus on the administration of unmodified donor bone marrow (BM) by either single or multiple infusions (7, 8). This is undertaken without recipient cytoablation. Conventional immunosuppressive therapy is believed to facilitate donor cell survival and to promote the reciprocal donor-recipient leukocyte engagement that may provide the basis of allograft acceptance (9). A potential chimerism-enhancing strategy that is well-recognized in BM transplantation, but that has not been evaluated formally in organ allograft recipients, is the use of hematopoietic growth factors. Many of these can either enhance, inhibit, or bi-functionally affect the proliferation and differentiation of hematopoietic progenitor cells (10–13). They comprise granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and a variety of other cytokines that act at an earlier point in the hematopoietic cascade, such as interleukin (IL) 3, stem cell factor (c-kit ligand), and the recently cloned flt3 ligand (FL) (14). In the hemopoietic system, the expression of flt3 (a member of the type III receptor tyrosine kinase family that also includes c-kit and fms) is believed to be restricted to primitive progenitor cells that respond to FL (.14–17) PL potently synergizes with the colony-stimulating factors and c-kit ligand to enhance growth of hemopoietic progenitors (14–19). Notably, there is recent evidence that in contrast to the case with GM-CSF, G-CSF, and c-kit ligand, administration of FL to normal mice leads to dramatic increases in DC numbers, both in lymphoid and nonlymphoid tissues (20). In this study, we examined the influence of short-term (7-day) FL treatment on donor leukocyte microchimerism, both in normal (noncytoablated) and tacrolimus (formerly FK506)-immunosuppressed recipients of unmodified allogeneic BM. For comparative purposes, similar experiments were undertaken with GM-CSF. The results show that when administered with tacrolimus, FL dramatically augments hematopoietic cell chimerism within recipient lymphoid tissue. We also show that donor major histocompatibility complex (MHC) class II+ cells with distinct DC characteristics are a prominent feature of the greatly enhanced donor cell population. These findings may have important implications for the therapeutic application of FL to augment chimerism and to promote donor-recipient leukocyte interactions that may predispose to improved organ allograft survival.

Hepatic grafts from non-heartbeating donors may alleviate the organ shortage, but they inherently suf­ fer from warm ischemia. In the present study, we tested our hypothesis that augmentation of endoge­ nous adenosine by inhibition of nucleoside transport with R75231 attenuates ischemic liver injury. Adult female beagle dogs underwent 2·hr hepatic vascular exclusion with venovenous bypass. R75231 was given to the animals by continuous intravenous infusion for 30 min before ischemia at a dose of 0.1 mg/kg (Group 2, n=6), 0.05 mg/kg (Group 3, n=6), or 0.025 mg/kg (Group 4, n=6). Nontreated animals were used as the control (Group 1, n=10). Animal survival, hepatic tissue blood tlow, liver function, and histopathology were ana­ lyzed. Two-week animal survival was 300/0 in Group 1, 88% in Group 2, 100% in Group 3, and 100% in Group 4. Postreperfusion hepatic tissue blood tlow was mark· edly improved by the treatment. Treatment signifi. cantly attenuated liver enzyme release, lipid peron­ dation, and changes in adenine nucleotides and purine catabolites. Structural abnormality of the liver after reperfusion was markedly improved by R75231 treatment, showing better architecture and less neu· trophil infiltration. Preischemic administration of a nucleoside transport inhibitor ameliorated ischemic liver injury due to the positive effects of augmented endogenous adenosine, and is applicable clinically when the liver is procured from a controlled non­ heartbeating donor. Liver ischemia causes progressive degradation of adeno­ sine triphosphate (ATP),* leading to an immense accumula­ tion of purine catabolites in ischemic tissues_ Adenosine, one of the intermediary products of the degradation cascade, has been known to exert various biological actions (5-10) such as increased blood flow, vasodilation, inhibition of free radical production, suppression of neutrophil activation, and preven­ tion of platelet aggregation. Upon reoxygenation, adenosine becomes an important substrate for ATP resynthesis. How­ ever, adenosine is rapidly deaminated into inosine during ischemia and transported out of the ischemic tissues after reperfusion, thereby depriving ischemic tissues of ade­ nosine's beneficial effects. In this study, in an attempt to establish a strategy for the use of livers from non-heartbea.t­ ing donors, we first tested our hypothesis that augmentation of endogenous adenosine by inhibiting its transport with a nucleoside transport inhibitor, R75231, attenuates warm ischemic liver injury, using a 2-hr total hepatic vascular exclusion model.