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Background: Terminal duct lobular units (TDLUs) are the anatomic sites of breast cancer initiation, and breast tissue involution resulting in lower TDLU counts has been associated with decreased breast cancer risk. The insulin-like growth factor (IGF) pathway plays a role in breast involution, and systemic changes in this developmental pathway occur with hypertensive disorders of pregnancy (HDP), which have also been associated with lower breast cancer risk, especially in women carrying a functional variant of IGF1R SNP rs2016347. We proposed that this breast cancer protective effect might be explained by increased breast tissue involution., Materials and Methods: We conducted a retrospective cohort study utilizing the Komen Tissue Bank, which collects breast tissue core biopsies from women without a history of breast cancer. Eighty white non-Hispanic women with a history of HDP were selected along with 120 nonexposed participants, and after genotyping for rs2016347, TDLU parameters were histologically measured blinded to participant characteristics from fixed biopsy sections., Results: Stratified models by HDP status demonstrated that among HDP+ participants, those carrying two T alleles of rs2016347 had a decrease in TDLU counts of 53.2% when compared to those with no T alleles ( p =0.049). Trend analysis demonstrated a multiplicative decrease in counts of 31.6% per T allele ( p =0.050). Although no statistically significant interaction was seen between HDP status and T alleles, interaction terms showed increasingly negative values reaching a p value of 0.124 for HDP × 2T alleles., Conclusions: The observed statistically significant decrease in TDLU counts signifies increased breast epithelial involution in women with prior HDP who inherited the TT genotype of IGF1R SNP rs2016347. The increasing degree of breast involution with greater rs2016347 T allele copy number is consistent with the known progressive reduction in IGF1R expression in breast and other normal tissues., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Mark J. Powell et al.)

Background: Microparticles are now recognised as true biological effectors with a role in immunopathology through their ability to disseminate functional properties. Diannexin, a homodimer of annexin V, binds to PS with a higher affinity and longer blood half-life than the monomer, inhibits prothrombinase complex activity thereby diminishing coagulation and reperfusion injury mediators and prevent microvesicle-mediated material transfer. Our aim was to determine if Diannexin could modulate microparticle production by endothelial cells by interacting with the phosphatidylserine exposure occurring during the release of these vesicles., Results: In this study we showed that fluorescently labelled Diannexin binds to calcimycin-activated endothelial cells but not to resting cells. After overnight incubation, Diannexin enters cells and their released MP carry Diannexin. Some Diannexin seems to be processed via early endosomes and later is found in lysosomes. Both unlabelled Diannexin and fluorescent Diannexin inhibit MP release from TNF-activated endothelial cells. However, Diannexin treatment does not prevent endothelial activation by TNF. In addition, the inhibitory effect of Diannexin on MP release could be observed when cells were pre-, concomitantly or post-treated with cytokines. Scanning electron microscopy showed differences in the numbers and types of protuberances at the cell surface when cells were treated or not with Diannexin. Finally, there is no apparent congruency between fluorescent Diannexin labelling and surface protuberances as shown by correlative microscopy., Conclusions: Altogether these data suggest that Diannexin can inhibit endothelial vesiculation by binding PS present either at the cell surface or at the level of the inner leaflet of the plasma membrane.

Background & Aims: Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis., Methods: C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function., Results: Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin., Conclusions: Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life., Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation., Methods: The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser-induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis., Results: Diannexin and annexin A5 bind PS with K(D) values of 0.6 and 5 nm, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nm (P<0.05-0.001 compared with control), and reduced platelet accumulation at 1 μg g(-1) (P<0.05) and activation at 0.25 μg g(-1) (P<0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g(-1) (P<0.01)., Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation., (© 2012 International Society on Thrombosis and Haemostasis.)

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this "suicidal emperipolesis" is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.

Introduction and Aims: In patients with ST-segment elevation myocardial infarction who receive percutaneous coronary intervention and stenting, a large zone with no-reflow is associated with adverse outcomes. During myocardial ischemia/reperfusion, phosphatidylserine (PS) translocates to the surface of endothelial cells triggering attachment of platelets and leukocytes, thus impairing microvascular blood flow. Diannexin, a recombinant dimer of the endogenous human annexin V protein, binds PS and thus inhibits the adverse effects of PS. It has been shown to attenuate postischemic reperfusion injury in several experimental models. We speculated that Diannexin would reduce no-reflow in the heart after coronary artery occlusion (CAO) and reperfusion. Rabbits received: (1) Diannexin 5 min pre-CAO (diannexin pre ischemia [DPI], 400 μg/kg, n = 17), or (2) Diannexin 5 min pre-coronary reperfusion (diannexin pre reperfusion [DPR], 400 μg/kg, n = 20), or (3) saline (Cont, n = 18), with 30 min CAO and 3 h reperfusion. In a secondary analysis, rabbits were divided into two groups based on the overall average risk zone size of 29% of the left ventricle (LV): small (<29% of LV) and large (>29% of LV)., Results: Overall, risk zones and infarct size, and the no-reflow zone were similar in all groups. In hearts with large risk zones the no-reflow area was significantly smaller in both drug-treated groups (DPI, 22 ± 5% and DPR, 22 ± 3% vs. control 40 ± 3%, P < 0.006), the hemorrhagic areas were significantly smaller, and infarct size was reduced at the P < 0.06 level compared with control. In animals with small risk zones there were no significant differences. Diannexin treatment did not affect hemodynamics or LV function., Conclusion: Diannexin was cardioprotective in rabbits with a severe ischemic insult. This is important, because large infarcts accompanied by no-reflow in humans are associated with increased complications. In animals with small risk zones, no significant drug effect was observed., (© 2010 Blackwell Publishing Ltd.)

Background: A major unmet challenge is to reduce the islet mass needed for insulin independence in type 1 diabetic recipients after islet transplantation. The recombinant homodimer of human annexin V, diannexin, has completed a Phase II Clinical Trial in Kidney Transplantation (NCT00615966)., Methods: We developed a marginal islet mass transplantation model (10-12 islets per gram of recipient body weight) and investigated whether diannexin prevents β-cell apoptosis and improves islet graft function. Diannexin was administered to islet cell donors shortly before pancreas harvest, added to isolation reagents, and infused into recipients at the time of transplantation and repeated daily until day 4., Results: In the syngeneic marginal islet mass transplantation model, the median time needed to achieve normoglycemia was reduced from 17.0 days among untreated controls to 3.5 days among diannexin-treated recipients (P=0.004). Histologic analysis of islet grafts harvested on day 3 posttransplantation revealed decreased macrophage (44.7%±9.8% vs. 19.2%±3.2%, P=0.007) and T-cell infiltration (25.9%±5.5% vs. 9.1%±1.1%, P=0.004), and a lower rate of islet cell apoptosis (20.5%±2.8% vs. 7.6%±2.3%, P=0.01) with diannexin treatment. Expression profiling of the islet grafts showed significantly lower levels of mRNA for the proapoptotic molecule Bid, but higher levels of interleukin-6, interferon-γ, and immunosuppressive cytokine interleukin-10., Conclusions: Our findings demonstrate that diannexin improves the early function of marginal mass islet grafts, and its effects are associated with reductions in inflammatory cell infiltration and β-cell death by apoptosis after islet transplantation.

Ischemia-reperfusion injury (IRI) is a common and serious complication of reperfusion following vascular occlusion. We present a novel interpretation of the pathogenesis of IRI. According to this hypothesis, anoxia resulting from ischemia allows translocation of phosphatidylserine to the surface of endothelial cells (ECs), providing an attachment site for leukocytes and platelets. This attachment impedes blood flow through the microvasculature. During IRI mediators of increased vascular permeability are produced, resulting in edema. We have developed a recombinant homodimer of human Annexin V, Diannexin, to attenuate IRI. Annexin V (36 kDa) rapidly passes from the circulation into the urine. In Diannexin 2 annexin V molecules are joined by a short peptide linker to produce a 73 kDa protein, which exceeds the renal filtration threshold. Diannexin has a half-life of about 2.5 hours in the human circulation. Diannexin also has a higher affinity for phosphatidylserine on cell surfaces than the monomer has. Such binding inhibits leukocyte attachment to ECs, and inflammatory mediator formation, during IRI. The aim of the study now reported was to ascertain the effects of Diannexin on IRI in the rat cremaster muscle flap, as revealed by intravital microscopy. During IRI there was increased attachment of leukocytes to ECs, reduced blood flow and augmented vascular permeability. Administration of Diannexin before or just after ischemia prevented these effects. Diannexin inhibited transmigration of leukocytes during IRI. Edema complicates peripheral vascular surgery, stroke, and other clinical conditions. Diannexin has proven to be safe when administered to patients after major surgical operations, and it may be useful to prevent IRI associated with peripheral vascular surgery.

The term 'innate resistance' covers mechanisms of resistance that operate early in the course of infections, preceding adaptive immune responses which exert effects after several days. The first example of genetically controlled innate resistance to human malaria was the demonstration in 1954 that sickle-cell heterozygotes have less severe Plasmodium falciparum infections than do children with normal adult hemoglobin. This observation has been repeatedly confirmed, most recently by independent studies of genome-wide associations in severe falciparum malaria, which have identified the HBB locus as the major signal of association. Other abnormal hemoglobins, glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency also confer some degree of resistance against falciparum malaria. A second early example of inherited innate resistance to malaria was the finding that nonexpression of the Duffy antigen/chemokine receptor (DARC) on erythrocytes confers resistance to P. vivax. However, this parasite can enter nonhuman primate red cells independently of DARC, and in some human populations P. vivax has been observed in persons lacking DARC. Hence DARC is not the only receptor for P. vivax, but it is likely to be a major one for human transmission. Innate resistance to malaria is rapidly reinforced by adaptive immune responses, both cell-mediated and humoral. Among the factors influencing the efficacy of adaptive immune responses to malaria is the MHC complex constitution of hosts. This differs among populations, presumably because of variations in the structure of parasite antigens recognized by the immune systems of hosts.

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenic endothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode.

The first phase of research on genetic factors influencing susceptibility to infectious diseases was observational and descriptive. It began with the identification of children and adults with selective and non-selective immunodeficiencies. The types of infections to which these patients are susceptible provided evidence for the roles of T-cells, B-cells, leukocytes, and complement in controlling infectious diseases. Later the biochemical bases for these deficiencies were characterized. For example, an abnormal tyrosine kinase is associated with X-linked agammaglobulinemia, and lack of adenosine deaminase results in severe combined immunodeficiency. The second strategy for analyzing inherited resistance to disease was hypothesis-driven. Observations on the distribution of the sickle-cell gene suggested that heterozygotes might be resistant to P. falciparum malaria. That proposal has been confirmed repeatedly. Persons heterozygous for other hemoglobin mutations and those with glucose-6-phosphate dehydrogenase deficiency also have some degree of resistance to malaria. The third, modern phase of research on susceptibility to infectious diseases is genomic. This powerful approach facilitated characterization of the mutations responsible for most of the above-mentioned defects. Moreover, genomics strategies make analyses of susceptibility to infections possible even when these are under multifactorial genetic control, as exemplified by malaria. This is likely to be true for most infectious diseases, so the genomic approach is an important way forward., (Copyright © 2008 S. Karger AG, Basel.)

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4 degrees C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 microg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-alpha, IL-1beta, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.

Background and Aims: Ischemia-reperfusion injury (IRI) remains an important cause of liver failure after hepatic surgery or transplantation. The mechanism seems to originate within the hepatic sinusoid, with damage to endothelial cells, an early, reproducible finding. Sinusoidal endothelial cells (SECs), damaged during reperfusion, activate and recruit inflammatory cells and platelets. We hypothesized that a recombinant human annexin V homodimer, Diannexin, would protect SECs from reperfusion injury., Methods: We tested this proposal in a well-characterized in vivo murine partial hepatic IRI model., Results: Whether administered 5 minutes or 24 hours before or 1 hour after ischemia-reperfusion, Diannexin (100-1000 microg/kg) almost completely protected against liver injury. The protective efficacy conferred by Diannexin was highly visible at the microcirculatory level. Thus, although IR in this model is associated with early swelling and gap formation in SECs, Diannexin ameliorated these effects as shown by >80% reduction in alanine aminotransferase values during the early phase of reperfusion injury (2 hours) and near normalization of liver necrosis and inflammation in the late phase of inflammatory recruitment (24 hours). Consistent with the proposed role of SEC injury in hepatic IRI, Diannexin also decreased hepatic expression of proinflammatory molecules (MIP-2, ICAM-1, VCAM), abolished leukocyte and platelet adherence to damaged SECs, and, by in vivo microscopy, Diannexin preserved microcirculatory blood flow and hepatocyte integrity during reperfusion., Conclusions: Diannexin is an apparently safe therapeutic protein that provides prolonged protection against hepatic IRI via cytoprotection of SECs, thereby interrupting secondary microcirculatory inflammation and coagulation.

Annexin V (AV), a protein with anticoagulant activity, exerts antithrombotic activity by binding to phosphatidylserine (PS), inhibiting activation of serine proteases important in blood coagulation. The potential use of this protein as an anticoagulant is limited as it rapidly passes from the blood into the kidneys due to its relatively small size (36 kDa). We used recombinant DNA technology to produce a homodimer of human AV (DAV, 73 kDa), which exceeds the renal filtration threshold, and has a 6.5-hour half-life in the rat circulation. Human red blood cells with externalized PS were used to show that DAV had a higher affinity for PS-exposing cells than AV. DAV labeling sensitively identifies PS-exposing cells, was found to be a potent inhibitor of the activity of the prothombinase complexes and inhibits the ability of secretory phospholipaseA(2) to hydrolyze phospholipids of PS-exposing cells, reducing the formation of mediators of blood coagulation and reperfusion injury. DAV exerts dose-dependent antithrombotic activity in rat veins. This combination of activities suggests that DAV is a valuable probe to measure PS exposure and may be efficacious as a novel drug in a wide range of clinical situations.

Atherosclerosis is characterized by a persistent, low-grade inflammatory state in which immune cell activation is inseparably linked to plaque formation and destabilization. The T-lymphocyte in particular has emerged as a pivotal player throughout the course of atherogenesis. As a consequence, the concept that immune modulation is a suitable target for cardiovascular prevention is currently an important focus of research. Mycophenolate mofetil (MMF) has emerged as a non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) that exerts cytostatic effects, particularly on proliferating T-lymphocytes. In addition, MMF has other immune-modulating effects, such as downregulation of the expression of adhesion molecules and attenuation of monocyte and macrophage responses. Given the added benefit that MMF is well tolerated, this immunosuppressive agent constitutes an attractive candidate for the modulation of inflammatory activation in atherogenesis. The present review provides an overview of the potential anti-atherogenic properties of MMF.

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), an inhibitor of inosine-5'-monophosphate dehydrogenase, has several immunosuppressant actions. MPA depletes guanosine and deoxyguanosine nucleotides preferentially in T and B lymphocytes, inhibiting proliferation and suppressing cell-mediated immune responses and antibody formation, major factors in acute and chronic rejection. MPA also can induce T-lymphocyte apoptosis. MPA suppresses dendritic cell maturation and can induce human monocyte-macrophage cell line differentiation, decreasing the expression of interleukin (IL)-1 and enhancing expression of the IL-1 receptor antagonist. In addition, MPA inhibits adhesion molecule glycosylation and expression and lymphocyte and monocyte recruitment. Activated macrophages produce nitric oxide (NO) and superoxide, which combine to generate tissue-damaging peroxynitrite. MPA depletes tetrahydrobiopterin and decreases NO production by inducible NO synthase without affecting constitutive NO synthase activity. By these mechanisms, MMF exerts anti-inflammatory activity, which could attenuate both acute and chronic rejection. Unlike calcineurin inhibitors, MMF is nonnephrotoxic and does not induce transforming growth factor-beta production, which is fibrogenic. MMF inhibits arterial smooth muscle cell proliferation, a contributor to graft proliferative arteriopathy, and does not increase blood pressure, cholesterol, or triglyceride levels. By decreasing high-density lipoprotein oxidation and macrophage recruitment, MMF also may delay onset/progression of graft atherosclerosis. Thus, MMF may prevent chronic rejection by several mechanisms. MMF activity is synergistic with that of other agents such as valganciclovir for treating cytomegalovirus infection. MMF also has synergistic activity with angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists in the treatment of some nephropathies in experimental animals. This combination may prevent progression toward end-stage renal disease in humans with chronic allograft, lupus, and diabetic nephropathies.

In the brains of patients with Alzheimer's disease (AD) signs of neuronal degeneration are accompanied by markers of microglial activation, inflammation, and oxidant damage. The presence of nitrotyrosine in the cell bodies of neurons in AD suggests that peroxynitrite contributes to the pathogenesis of the disease. A drug with antioxidant and anti-inflammatory activity may prevent neuronal degeneration in AD. Celastrol, a plant-derived triterpene, has these effects. In low nanomolar concentrations celastrol was found to suppress the production by human monocytes and macrophages of the pro-inflammatory cytokines TNF-alpha and IL-1beta. Celastrol also decreased the induced expression of class II MHC molecules by microglia. In macrophage lineage cells and endothelial cells celastrol decreased induced but not constitutive NO production. Celastrol suppressed adjuvant arthritis in the rat, demonstrating in vivo anti-inflammatory activity. Low doses of celastrol administered to rats significantly improved their performance in memory, learning and psychomotor activity tests. The potent antioxidant and anti-inflammatory activities of celastrol, and its effects on cognitive functions, suggest that the drug may be useful to treat neurodegenerative diseases accompanied by inflammation, such as AD.

The incidence of Alzheimer's disease (AD) increases with age and in carriers of the apolipoprotein E4 genotype. A relative deficiency of vitamin K, affecting the extrahepatic functions of the vitamin, is common in ageing men and women. The concentration of vitamin K is lower in the circulating blood of APOE4 carriers than in that of persons with other APOE genotypes. Evidence is accumulating that vitamin K has important functions in the brain, including the regulation of sulfotransferase activity and the activity of a growth factor/tyrosine kinase receptor (Gas 6/Axl). The hypothesis is now proposed that vitamin K deficiency contributes to the pathogenesis of AD and that vitamin K supplementation may have a beneficial effect in preventing or treating the disease. Vitamin K may also reduce neuronal damage associated with cardiovascular disease., (Copyright 2001 Harcourt Publishers Ltd.)

The aim of our research was to develop a quantitative assay for angiogenesis in mammals, especially the mouse. This is a convenient experimental animal because of its small size, which allows compact housing and experimentation with angiogenic factors or inhibitors in limited supply. Mouse genetics is an advanced discipline, resulting in the availability of many inbred strains and histocompatible tumors. Recombinant growth factors and other proteins are usually of human or mouse origin, and the desirability of using proteins of the experimental animal under study has been demonstrated. Mice genetically engineered to overproduce or not produce particular growth factors or receptors are valuable experimental tools.

During the past 50 years, many immunosuppressive drugs have been described. Often their mechanisms of action were established long after their discovery. Eventually these mechanisms were found to fall into five groups: (i) regulators of gene expression; (ii) alkylating agents; (iii) inhibitors of de novo purine synthesis; (iv) inhibitors of de novo pyrimidine synthesis; and (v) inhibitors of kinases and phosphatases. Glucocorticoids exert immunosuppressive and anti-inflammatory activity mainly by inhibiting the expression of genes for interleukin-2 and other mediators. Cyclophosphamide metabolites alkylate DNA bases and preferentially suppress immune responses mediated by B-lymphocytes. Methotrexate and its polyglutamate derivatives suppress inflammatory responses through release of adenosine; they suppress immune responses by inducing the apoptosis of activated T-lymphocytes and inhibiting the synthesis of both purines and pyrimidines. Azathioprine metabolites inhibit several enzymes of purine synthesis. Mycophenolic acid and mizoribine inhibit inosine monophosphate dehydrogenase, thereby depleting guanosine nucleotides. Mycophenolic acid induces apoptosis of activated T-lymphocytes. A leflunomide metabolite and Brequinar inhibit dihydroorotate dehydrogenase, thereby suppressing pyrimidine nucleotide synthesis. Cyclosporine and FK-506 (Tacrolimus) inhibit the phosphatase activity of calcineurin, thereby suppressing the production of IL-2 and other cytokines. In addition, these compounds have recently been found to block the JNK and p38 signaling pathways triggered by antigen recognition in T-cells. In contrast, rapamycin inhibits kinases required for cell cycling and responses to IL-2. Rapamycin also induces apoptosis of activated T-lymphocytes. Immunosuppressive and anti-inflammatory compounds in development include inhibitors of p38 kinase and of the type IV isoform of cyclic AMP phosphodiesterase which is expressed in lymphocytes and monocytes.A promising future application of immunosuppressive drugs is their use in a regime to induce tolerance to allografts. The role of leukocytes in grafts, and the induction of apoptosis of clones of responding T-lymphocytes, is discussed.

Mycophenolate mofetil (MMF, CellCept(R)) is a prodrug of mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). This is the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. T- and B-lymphocytes are more dependent on this pathway than other cell types are. Moreover, MPA is a fivefold more potent inhibitor of the type II isoform of IMPDH, which is expressed in activated lymphocytes, than of the type I isoform of IMPDH, which is expressed in most cell types. MPA has therefore a more potent cytostatic effect on lymphocytes than on other cell types. This is the principal mechanism by which MPA exerts immunosuppressive effects. Three other mechanisms may also contribute to the efficacy of MPA in preventing allograft rejection and other applications. First, MPA can induce apoptosis of activated T-lymphocytes, which may eliminate clones of cells responding to antigenic stimulation. Second, by depleting guanosine nucleotides, MPA suppresses glycosylation and the expression of some adhesion molecules, thereby decreasing the recruitment of lymphocytes and monocytes into sites of inflammation and graft rejection. Third, by depleting guanosine nucleotides MPA also depletes tetrahydrobiopterin, a co-factor for the inducible form of nitric oxide synthase (iNOS). MPA therefore suppresses the production by iNOS of NO, and consequent tissue damage mediated by peroxynitrite. CellCept(R) suppresses T-lymphocytic responses to allogeneic cells and other antigens. The drug also suppresses primary, but not secondary, antibody responses. The efficacy of regimes including CellCept(R) in preventing allograft rejection, and in the treatment of rejection, is now firmly established. CellCept(R) is also efficacious in several experimental animal models of chronic rejection, and it is hoped that the drug will have the same effect in humans.

Microfluidized squalene or squalane emulsions are efficient adjuvants, eliciting both humoral and cellular immune responses. Microfluidization stabilizes the emulsions and allows sterilization by terminal filtration. The emulsions are stable for years at ambient temperature and can be frozen. Antigens are added after emulsification so that conformational epitopes are not lost by denaturation and to facilitate manufacture. A Pluronic block copolymer can be added to the squalane or squalene emulsion. Soluble antigens administered in such emulsions generate cytotoxic T lymphocytes able to lyse target cells expressing the antigen in a genetically restricted fashion. Optionally a relatively nontoxic analog of muramyl dipeptide (MDP) or another immunomodulator can be added; however, the dose of MDP must be restricted to avoid systemic side effects in humans. Squalene or squalane emulsions without copolymers or MDP have very little toxicity and elicit potent antibody responses to several antigens in nonhuman primates. They could be used to improve a wide range of vaccines. Squalene or squalane emulsions have been administered in human cancer vaccines, with mild side effects and evidence of efficacy, in terms of both immune responses and antitumor activity., (Copyright 1999 Academic Press.)

Background: Modulation of cytokine secretion may be of interest in the treatment of Crohn's disease or ulcerative colitis., Methods: The effect of three antioxidants - butylated hydroxyanisol, tetrahydropapaveroline and nordihydroguaiaretic acid - on the production of tumour necrosis factor (TNF), interleukin (IL) 1, IL-6 and IL-8 (measured by enzyme-linked immunosorbent assay) by peripheral mononuclear cells and biopsies of inflamed colonic mucosa from inflammatory bowel disease patients were studied., Results: We observed a decrease in IL-1 and IL-6 production by peripheral mononuclear cells from inflammatory bowel disease patients (approximately 50% of control). The three drugs did not decrease IL-6 and IL-8 secretion by colonic biopsies, whereas they did inhibit IL-1 and, to some degree, TNF production. The cytokine-inhibitory effect of antioxidants seems to be more pronounced in ulcerative colitis than in Crohn's disease., Conclusion: Our results suggest that the studied antioxidants, or related compounds, may be of interest in inflammatory bowel disease treatment.

We have previously described inhibition of the synthesis of three acute-phase inflammatory cytokines in human and rat macrophages by acetate esters of rooperol, a dicatechol of plant origin. Analysing the mechanism of anticytokine activity of rooperol, we compared levels of TNFalpha, IL-1beta and IL-6 mRNAs in the human promonocytic U937 cell line pretreated with phorbol myristate acetate (PMA) and incubated with rooperol tetraacetate (RTA) alone or in combination with LPS (500 ng/ml). It was found that 10 microM RTA decreased the levels of cytokine mRNAs both in the presence and absence of LPS, suggesting pretranslational inhibition of cytokine synthesis. Electrophoretic mobility shift analysis (EMSA) showed that RTA may influence cytokine mRNA expression by decreasing the binding activity of transcription factors NF-kappaB and AP-1.

Adjuvants augment immune responses to antigens and influence the balance between cell-mediated and humoral responses, as well as the isotypes of antibodies formed. New adjuvant formulations include antigen-carrying vehicles and small molecules with immunomodulating activity. Widely used two-phase vehicles comprise liposomes and microfluidized squalene or squalane emulsions. These are believed to target antigens to antigen-presenting cells, including dendritic cells (DC), follicular dendritic cells (FDC) and B-lymphocytes. Activation of complement generates C3d, which binds CR2 (CD21) on FDC and B-lymphocytes, thereby stimulating the proliferation of the latter and the generation of B-memory. Targeting of antigens to DC may favour cell-mediated immunity. Immunomodulating agents induce the production of cytokine cascades. In a primary cascade at injection sites TNF-alpha, GM-CSF and IL-1 are produced. TNF-alpha promotes migration of DC to lymphoid tissues, while GM-CSF and IL-1 accelerate the maturation of DC into efficient antigen-presenting cells for T-lymphocytes. In a secondary cytokine cascade in draining lymph nodes, DC produce IL-12, which induces Th1 responses with the production of IFN-gamma. The cytokines elicit cell-mediated immune responses and the formation of antibodies of protective isotypes, such as IgG2a in the mouse and IgG1 in humans. Antibodies of these isotypes activate complement and collaborate with antibody-dependent effector cells in protective immune responses.

Induced expression of vascular cell adhesion molecule-1 (VCAM-1) and of nitric oxide synthase (iNOS) is believed to play a role in the pathogenesis of atherosclerosis, asthma, as well as other inflammatory disorders. In the current study we examined the effect of the di-catechol rooperol [(E)-1,5-bis (3',4'-dihydroxyphenyl) pent-4-en-1-yne] on the process of microvascular endothelial cell (MME) activation by TNF-alpha and IFN-gamma. We show that rooperol decreases VCAM-1 and iNOS mRNA levels in cytokine-activated MME with subsequent inhibition of VCAM-1 membrane expression as measured by adhesion of P815 cells to MME monolayers, and NO production, as reflected in the nitrite concentration in culture medium. The properties of rooperol now described suggest that rooperol may be an anti-inflammatory agent useful in the treatment of several inflammatory disorders.

Luminol-enhanced chemiluminescence was measured in fresh whole human blood, or human neutrophils isolated from heparinized blood, human alveolar macrophages and rat alveolar macrophages stimulated with bacterial endotoxin (LPS). Tetraacetate esters of rooperol, a dicatechol showing anticytokine activity, added to cells simultaneously with LPS inhibited the respiratory burst. The effective concentrations of rooperol were in the range of 1-10 muM depending on cell type and corresponded well with inhibition of nitric oxide production by rat alveolar macrophages. Thus rooperol may reduce some effects of excessive phagocytic activity and inflammatory reaction but by quenching free radicals production may also diminish the resistance to bacterial infections.

New adjuvant formulations contain a vehicle, which carries antigens to antigen-presenting cells. Examples of vehicles are liposomes, immune-stimulating complexes and microfluidized squalene-in-water emulsions. Adjuvant formulations may contain immunomodulators, which augment cytokine production, such as a synthetic muramyl dipeptide analog or monophosphoryl lipid A. In a primary cascade of cytokine production at the site of antigen + adjuvant injection, TNF-alpha promotes the migration of dendritic cells (DC) to lymphoid tissues while GM-CSF accelerates the differentiation of DC into efficient presenters of antigens to T cells. Adjuvants also up-regulate a secondary cascade of cytokines in lymphoid tissues responding to antigenic stimulation: IL-12 augments the production of IFN-gamma, which favors the production of antibodies of protective isotypes (IgG2a in the mouse). Thus adjuvants can regulate immune responses qualitatively as well as quantitatively. Adjuvant formulations can also activate complement, generating C3d, which binds CD21 on follicular dendritic cells (FDC) and B cells. FDC targeting favors the generation of B lymphocyte memory, which is important for vaccination.

The ability of dicatechol rooperol and esters to inhibit the production of cytokines in endotoxin-stimulated human alveolar macrophages, human blood monocyte/macrophages, histiocytic cell line U937, and rat alveolar macrophages was examined in vitro. Rooperol derivatives inhibited the production of tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. Of the esters tested on human cells, rooperol diacetate and tetraacetate were more potent inhibitors of cytokine production (IC50 in the range of 10-20 microM) than rooperol disulphate (IC50 in the range of 25-75 microM). The acetate esters also inhibited cytokine production in rat alveolar macrophages, whereas the sulphate had little effect. Rooperol and acetate esters, in the same concentration range, decreased the production of nitric oxide by rat alveolar macrophages stimulated by endotoxin. These concentrations of rooperol had no effect on cell viability, as indicated by incorporation of 14C-labelled leucine into macrophage proteins and their content of lactate dehydrogenase. The results obtained suggest that rooperol esters are potentially useful antiinflammatory agents.

Summary: The true role of transforming growth factor beta1 (TGFbeta) on angiogenesis is in question. Several in vitro studies have shown inhibition of proliferation and migration of endothelial cells (EC). However, some investigators have observed that TGFbeta stimulates the formation of EC tubes in vitro. Fewer in vivo studies have been performed, but these also show discrepancies: some report angiogenic induction and at least one reports inhibition. We used the disc angiogenesis system (DAS) to measure the in vivo effect of TGFbeta. Discs containing 1 ng to 2000 ng of TGFbeta were placed subcutaneously in mice, removed after a growth period of 14 days, measured by three different techniques, and compared with spontaneous growth controls and with positive controls containing prostaglandin El. Tritiated thymidine was used to determine proliferation of EC. The discs were also examined morphologically for patterns of vessel and stromal proliferation. The contribution of native TGFbeta to the spontaneous angiogenesis of wound healing was tested using a monoclonal anti-TGFbeta antibody. The combined effect of TGFbeta and fibroblast growth factor (bFGF) was studied by using suboptimal doses of both (500 ng and 10 microg, respectively). Although TGFbeta doses of 1 ng to 500 ng failed to induce angiogenesis, 1000 ng induced a significant level of angiogenesis which was maintained at 2000 ng. This effect was the same regardless of the method of quantification: centripetal growth of the vessels, size of fibrovascular growth area, or amount of incorporation of tritiated thymidine. The anti-TGFbeta antibody decreased the spontaneous vascular growth below the level of controls containing irrelevant IgG. The combination of TGFbeta and bFGF at suboptimal doses did not increase or decrease the angiogenic response. Discs containing TGFbeta showed more collagen and greater accumulation of neutrophils than control discs or discs containing other cytokines. In conclusion, TGFbeta1 is angiogenic in vivo, when it reaches a threshold of 1 microg, but is not angiogenic at doses of 1 to 500 ng. Endogenous TGFbeta contributes to spontaneous (wound healing) angiogenesis. At the suboptimal doses of TGFbeta and bFGF used, there is no evidence of a combined angiogenic effect. The angiogenic effect of TGFbeta is probably indirect, requiring recruitment of leukocytes that secondarily release angiogenic substances. This secondary effect may explain some of the discrepancies between the in vitro and in vivo effects of TGFbeta.

Cytokines have been implicated in the regulation of antibody and inflammatory responses, but their role in periodontal diseases has not been elucidated. In the present study, cytokine production by human gingival fibroblasts (HGF) following in vitro passage was assessed in order to determine the basal levels of cytokine message and protein and to determine if the cellular morphology and the profile of cytokines produced differed with passage. The HGF cell line F-CL was established by explantation from non-inflamed gingival tissue, and cells from passages 1-10 were studied. The number of cells was determined in confluent cultures and cell morphology was examined by light microscopy. Fibroblasts from confluent cultures were examined for cytokine mRNA by reverse transcription-polymerase chain reaction and culture supernatants were assessed for cytokines by enzyme-linked immunosorbent assay. The morphology of F-CL fibroblasts in passages 1-4 was normal, while fibroblasts in passages 5-10 were larger. In general, the number of cells decreased from early to late passage. Fibroblasts from passages 1-10 contained message for interleukin-1 beta, -6 and -8, but not for interleukin-1 alpha or tumour necrosis factor-alpha. Interleukin-6 was detected in culture supernatants of F-CL fibroblasts by the enzyme immunoassay and its level decreased with increasing passage.

Mycophenolate mofetil (MMF) is a novel immunosuppressive drug that shows promise in preventing the rejection of organ allografts and in the treatment of ongoing rejection. Orally administered MMF is hydrolyzed by esterases in the intestine and blood to release mycophenolic acid (MPA), a potent, selective, noncompetitive inhibitor of the type 2 isoform of inosine monophosphate dehydroxygenase (IMPDH) expressed in activated human T and B lymphocytes. By inhibiting IMPDH, MPA depletes the pool of dGTP required for DNA synthesis. MPA has a more potent cytostatic effect on lymphocytes than on other cell types, and this is the principal mechanism by which immunosuppressive activity is exerted. MPA also depletes pools of GTP in human lymphocytes and monocytes, thereby inhibiting the synthesis of fucose- and mannose-containing saccharide components of membrane glycoproteins. These are recognized by the family of adhesion molecules termed selectins. By this mechanism, MPA could decrease the recruitment of lymphocytes and monocytes into sites of graft rejection. In addition to preventing allograft rejection, MMF suppresses graft-versus-host reactions in lethal and nonlethal murine models. MMF inhibits primary antibody responses more efficiently than secondary responses. MPA inhibits the proliferation of human B lymphocytes transformed by Epstein-Barr virus and is not mutagenic. Clinically attainable concentrations of MPA suppress the proliferation of human arterial smooth muscle cells. These two properties of MPA may decrease the risk of lymphoma development and proliferative arteriopathy in long-term recipients of MMF.

Mycophenolate mofetil (MM), an inhibitor of purine metabolism, was found to effectively inhibit the development of experimental autoimmune uveoretinitis (EAU) induced by S-antigen (SAg) in Lewis rats. MM completely inhibited EAU development in the majority of rats when administered daily, on days 0-13, at a dose of 30 mg kg-1 day-1. The drug was less effective, however, when given on days 7-20: minimal disease inhibition was achieved with the drug at 30 mg kg-1 day-1, although at 60 mg kg-1 day-1 the drug inhibited EAU development in most treated rats during the period of its administration. MM also completely inhibited in most treated rats the development of EAU adoptively transferred by SAg-sensitized lymphocytes, thus depicting its capacity to inhibit the efferent limb of the immune response. Treatment with MM also suppressed the cellular and humoral immune responses against SAg, with a good correlation being observed between the inhibition of these responses and suppression of EAU in the different groups of rats. MM is currently being examined for its immunosuppressive effects in humans and the data recorded here thus suggest this compound may be useful in treatment of immune-mediated uveitic conditions.