Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38: Implications for Cellular NAD+ Metabolism,Protein Acetylation,and Treatment of Metabolic Syndrome

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD⁺ metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD⁺ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD⁺ase in mammals. Moreover, CD38 knockout mice have higher NAD⁺ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD⁺ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD⁺ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD⁺ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD⁺-dependent pathways.Obesity is a disease that has reached epidemic proportions in developed and developing countries (1–3). In the U.S., >60% of the population is overweight (1,3,4). Obesity is a feature of metabolic syndrome, which includes glucose intolerance, insulin resistance, dyslipidemia, and hypertension. These pathologies are well-documented risk factors for cardiovascular disease, type 2 diabetes, and stroke (4). It is therefore imperative to envision new strategies to treat metabolic syndrome and obesity.Recently, the role of NAD⁺ as a signaling molecule in metabolism has become a focus of intense research. It was shown that an increase in intracellular NAD⁺ levels in tissues protects against obesity (5,6), metabolic syndrome, and type 2 diabetes (5–7). Our group was the first to demonstrate that an increase in NAD⁺ levels protects against high-fat diet–induced obesity, liver steatosis, and metabolic syndrome (5). This concept was later expanded by others using different approaches, including inhibition of poly-ADP-ribose polymerase (PARP)1 (6) and stimulation of NAD⁺ synthesis (7).The ability of NAD⁺ to affect metabolic diseases seems to be mediated by sirtuins (8). This family of seven NAD⁺-dependent protein deacetylases, particularly SIRT1, SIRT3, and SIRT6, has gained significant attention as candidates to treat metabolic syndrome and obesity (9). Sirtuins use and degrade NAD⁺ as part of their enzymatic reaction (8), which makes NAD⁺ a limiting factor for sirtuin activity (9). In particular, silent mating information regulation 2 homolog 1 (SIRT1) has been shown to deacetylate several proteins, including p53 (10), RelA/p65 (11), PGC1-α (12), and histones (13), among others. In addition, increased expression of SIRT1 (14), increased SIRT1 activity (15), and pharmacological activation of SIRT1 (16) protect mice against liver steatosis and other features of metabolic syndrome when mice are fed a high-fat diet. Given the beneficial consequences of increased SIRT1 activity, great efforts are being directed toward the development of pharmacological interventions aimed at activating SIRT1.We previously reported that the protein CD38 is the primary NAD⁺ase in mammalian tissues (17). In fact, tissues of mice that lack CD38 contain higher NAD⁺ levels (17,18) and increased SIRT1 activity compared with wild-type mice (5,17). CD38 knockout mice are resistant to high-fat diet–induced obesity and other aspects of metabolic disease, including liver steatosis and glucose intolerance, by a mechanism that is SIRT1 dependent (5). These multiple lines of evidence suggest that pharmacological CD38 inhibition would lead to SIRT1 activation through an increase in NAD⁺ levels, resulting in beneficial effects on metabolic syndrome.Recently, it was shown that in vitro, CD38 is inhibited by flavonoids, including quercetin (19). Flavonoids are naturally occurring compounds present in a variety of plants and fruits (20). Among them, quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one] and apigenin [5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] have been shown to have beneficial effects against cancer (21–24). In fact, apigenin and quercetin ameliorate atherosclerosis (25) and reduce inflammation (26–28). However, the mechanisms of action of flavonoids remain largely unknown. We hypothesized that the effect of some flavonoids in vivo may occur through inhibition of CD38 and an increase in NAD⁺ levels in tissues, which lead to protection against metabolic syndrome.Here, we show that CD38 expression and activity regulate cellular NAD⁺ levels and global acetylation of proteins, including SIRT1 substrates. We confirmed that quercetin is a CD38 inhibitor in vitro and in cells. Importantly, we demonstrate that apigenin is a novel inhibitor of CD38 in vitro and in vivo. Treatment of cells with apigenin or quercetin inhibits CD38 and promotes an increase in intracellular NAD⁺ levels. An increased NAD⁺ level decreases protein acetylation through sirtuin activation. Finally, treatment of obese mice with apigenin results in CD38 inhibition, higher NAD⁺ levels in the liver, and a decrease in protein acetylation. Apigenin treatment improves glucose homeostasis, glucose tolerance, and lipid metabolism in obese mice. Our results clearly demonstrate that CD38 is a novel therapeutic target for the treatment of metabolic diseases and that apigenin and quercetin as well as other CD38 inhibitors may be used to treat metabolic syndrome.     

Aldose Reductase Drives Hyperacetylation of Egr-1 in Hyperglycemia and Consequent Upregulation of Proinflammatory and Prothrombotic Signals

Sustained increases in glucose flux via the aldose reductase (AR) pathway have been linked to diabetic vascular complications. Previous studies revealed that glucose flux via AR mediates endothelial dysfunction and leads to lesional hemorrhage in diabetic human AR (hAR) expressing mice in an apoE^−/− background. Our studies revealed sustained activation of Egr-1 with subsequent induction of its downstream target genes tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in diabetic apoE^−/−hAR mice aortas and in high glucose–treated primary murine aortic endothelial cells expressing hAR. Furthermore, we observed that flux via AR impaired NAD⁺ homeostasis and reduced activity of NAD⁺-dependent deacetylase Sirt-1 leading to acetylation and prolonged expression of Egr-1 in hyperglycemic conditions. In conclusion, our data demonstrate a novel mechanism by which glucose flux via AR triggers activation, acetylation, and prolonged expression of Egr-1 leading to proinflammatory and prothrombotic responses in diabetic atherosclerosis.     

Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD⁺) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD⁺ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m²) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD⁺ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD⁺ boosters can also directly affect skeletal muscle mitochondrial function in humans.     

Bacteremic shock: aspects of high-energy metabolism of rat liver following living Escherichia coli injection

Sublethal and lethal doses of living Escherichia coli (E. coli) (serotype: O-18) were injected intravenously into rats. The amounts of live E. coli injected were 0.5 ml of 0.8 – 1.0 × 10⁹ or 2.5 – 3.0 × 10⁹ organisms/ml saline per 100 g body weight for the sublethal (SL) or lethal (L) groups, respectively. The adenylate energy charge (ATP + 1/2ADP/ATP + ADP + AMP) and free NAD⁺/NADH ratios of the mitochondrial and cytoplasmic compartments in rat livers were measured at 3, 6, 12, and 24 hr following E. coli injection. At 6 hr in both groups, the hepatic energy charge levels were decreased from 0.856 in controls to 0.823 (SL) and 0.812 (L), associated with elevated mitochondrial NAD⁺/NADH ratios (P < 0.05). At 12 hr the sublethal group maintained the above state. In the lethal group, however, the hepatic energy charge was markedly decreased (P < 0.001), associated with remarkably decreased NAD⁺/NADH ratios in both mitochondria and cytoplasm (P < 0.01 and P < 0.001). In both groups the total ketone body (acetoacetate + β-hydroxybutyrate) contents were decreased with an elevation of mitochondrial NAD⁺/NADH. From these results, it is concluded that the difference between the sublethal and lethal groups is characterized by the progressive deterioration of the high-energy level in the lethal group, possibly due to restricted mitochondrial respiration. In addition, it is suggested that the elevated mitochondrial NAD⁺/NADH ratio may play a role in decreased ketone body contents in the liver following E. coli injection.     

PARP-1 Inhibits Glycolysis in Ischemic Kidneys

After ischemic renal injury (IRI), selective damage occurs in the S₃ segments of the proximal tubules as a result of inhibition of glycolysis, but the mechanism of this inhibition is unknown. We previously reported that inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) activity protects against ischemia-induced necrosis in proximal tubules by preserving ATP levels. Here, we tested whether PARP-1 activation in proximal tubules after IRI leads to poly(ADP-ribosyl)ation of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a modification that inhibits its activity. Using in vitro and in vivo models, under hypoxic conditions, we detected poly(ADP-ribosyl)ation and reduced activity of GAPDH; inhibition of PARP-1 activity restored GAPDH activity and ATP levels. Inhibition of GAPDH with iodoacetate exacerbated ATP depletion, cytotoxicity, and necrotic cell death of LLCPK₁ cells subjected to hypoxic conditions, whereas inhibition of PARP-1 activity was cytoprotective. In conclusion, these data indicate that poly(ADP-ribosyl)ation of GAPDH and the subsequent inhibition of anaerobic respiration exacerbate ATP depletion selectively in the proximal tubule after IRI.Compromised perfusion of renal tissues leading to ischemic renal injury (IRI), generally accepted as the major cause of acute kidney injury (AKI), usually results from hypoxia-induced renal vascular and tubular dysfunction. The outer medullary region of the kidney receives <10% of the blood delivered to the kidney via the renal artery. After IRI, a persistent perfusion deficit exists even at 24 h after reperfusion, and the outer medullary partial pressure of oxygen is restored to only 10% of its normal levels, rendering this region susceptible to injury at both the tubular and vascular levels.^1–3 Thus, the prolonged perfusion deficit shuts down oxidative phosphorylation in the cells of the outer medullary segments of the nephron and reverts to anaerobic metabolism for ATP synthesis. Nevertheless, ischemia causes selective injury to the outer medullary proximal straight tubules (PST), causing the PST cells to undergo cell death and/or sublethal injury to instigate renal dysfunction.^4,5 The medullary thick ascending limb, although situated in the same region, does not undergo injury to the same level.^6–8 Despite ongoing debate for more than two decades, the molecular mechanisms by which PST cells undergo selective injury are not known.Hypoxia resulting from decreased blood flow leads to a variety of secondary effects, including a breakdown in cellular energy metabolism and generation of reactive oxygen species (ROS) and reactive nitrogen species.^9,10 The superoxides induce DNA strand breaks in ischemic kidneys as early as 1 h after IRI.¹¹ The severe DNA damage that ensues results in excessive activation of the DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1), which exacerbates ATP depletion and triggers signaling cascades, leading to cellular suicide.¹² Recent data from our laboratory showed selective upregulation of PARP-1 expression and its activity in PST cells after renal ischemia.^13,14 Gene ablation or pharmacologic inhibition of PARP-1 activity offers both functional and histopathologic protection from IRI.^13,15 The ATP levels are significantly preserved in both in vivo and in vitro models of IRI after PARP gene ablation or inhibition, respectively^13,15,16; however, the exact mechanisms by which PARP activation leads to ATP depletion and whether these mechanisms are linked to selective damage to PST after renal ischemia are not defined.According to the “cell suicide” hypothesis, PARP-1 activation induces energy failure by depleting NAD⁺, and the cell consumes ATP to replete the NAD⁺ level, ultimately leading to energy failure and cell death¹²; however, the role of NAD⁺ in energy depletion is controversial, and its depletion alone may not be lethal to cells.^17,18 Moreover, PST cells can carry out anaerobic respiration under hypoxic conditions and be protected from injury. Nevertheless, under anoxic conditions, significant amounts of ATP are generated by anaerobic glycolysis by thick ascending limbs but not by the proximal tubular cells.¹⁹ These findings prompted us to investigate whether PARP activation interferes with glycolytic ATP synthesis and thus exacerbates ATP depletion and PST cell injury.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway and is susceptible to several modifications that alter its activity, including oxidative modification of thiols and mono-ADP-ribosylation.^20–22 Recently, PARP-1 was reported to inhibit GAPDH activity by poly(ADP-ribosyl)ation after hyperglycemia-induced aortic endothelial cell injury.²³ The temporal and spatial expression pattern of PARP-1 in PST during the time of ischemic injury prompted us to hypothesize that poly(ADP-ribosyl)ation and inhibition of the GAPDH leads to inhibition of glycolysis, reducing ATP synthesis and exacerbating energy depletion and cell injury.In this study, we evaluated the role of GAPDH-poly(ADP-ribosyl)ation as a mechanism to inhibit GAPDH activity in PST after IRI using in vitro and in vivo models. We explored the role of GAPDH-poly(ADP-ribosyl)ation in inhibiting glycolysis, exacerbating ATP depletion, and inducing cell death. Our findings suggest that PARP-1–mediated anaerobic glycolytic inhibition is a key mechanism of selective PST injury after IRI.     

The In Vitro Protection of Human Decay Accelerating Factor and hDAF/Heme Oxygenase-1 Transgenes in Porcine Aortic Endothelial Cells Against Sera of Formosan Macaques

To mitigate hyperacute rejection, pigs have been generated with α-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF⁺, hDAF⁺/hHO-1⁺, and hDAF⁺/hHO-1⁻ and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF⁺/hHO-1⁺ showed no further protection against effects of MS on transgenic pAEC.     

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline–alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na⁺-Cl⁻ cotransporter (NCC) and Na⁺-K⁺-2Cl⁻ cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.     

Can adenine nucleotides predict primary nonfunction of the human liver homograft?

Sixty-eight primary liver grafts were analyzed to see whether adenine nucleotides (AN: ATP, ADP, and AMP) or purine catabolites (PC: adenosine, inosine, hypoxanthine, and xanthine) of tissue or effluent can predict primary graft nonfunction. AN, PC, and nicotinamide adenine dinucleotide, oxidized form (NAD⁺) of the tissue before (pretransplant) and after graft reperfusion (post-transplant) and of the effluent were analyzed. The graft outcome was classified into two groups (group A: successful, n=64; group B: primary nonfunctioning, n=4). No significant differences were observed in pretransplant measurements between groups A and B, whereas ATP, ADP, total AN, total AN+total PC (T) and NAD⁺, in post-transplant tissues, were significantly higher in group A. Xanthine in the effluent was significantly higher in group B than in group A. ATP, ADP, total AN, T, and NAD⁺ in post-transplant tissue were significantly associated with primary graft nonfunction by logistic regression analysis.     

Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3‐Kinase and MEK Pathways

Phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase/extracellular signal‐regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4⁺ and CD8⁺ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4⁺ and CD8⁺ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4⁺ and CD8⁺ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity.     

Pathomechanismen des Organversagens

Proinflammatory mediators as well as increased formation of reactive oxygen and nitrogen species impair cellular respiration during sepsis. In particular, the highly reactive peroxynitrite irreversibly damages lipids, proteins and nucleic acids and also inhibits enzyme complexes of the respiratory chain. In this way cellular metabolic functions and subsequently organ functions are also impaired. Repair of DNA by poly(ADP-ribose)polymerase consumes large amounts of nicotinamide adenine dinucleotide (NAD⁺) which leads to cellular NAD⁺ depletion further promoting inflammation. This article summarizes central aspects of the pathophysiology of mitochondrial dysfunction during sepsis and gives an overview about newly developed strategies which proved effective in experimental studies and may have a potential clinical application in the future.     

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4+ memory T cells in mice

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4⁺ Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5 days to 9 days in recipient mice with CD4⁺ Tm infusion (P < 0.01), which however was not significantly longer than that (8 days) in untreated recipient mice without CD4⁺ Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4⁺ Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4⁺ Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44^highCD62L⁻ effector/memory as well as CD69⁺/CD25⁺ activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4⁺ Tm, for serum level of IFN-γ and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4⁺ Tm cells responding to the alloantigen, indicating that alloreactive CD4⁺ Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4⁺ Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.     

Effects of imiquimod and low-intensity laser (λ660nm) in chemically induced oral carcinomas in hamster buccal pouch mucosa

Squamous cell carcinoma (SCC) is the most common neoplasm of the oral cavity. It is aggressive, highly proliferative, and metastatic. This study aimed to evaluate the effect of LLLT and imiquimod on DMBA chemically induced lesions on the oral mucosa of hamsters. SCCs were induced on 25 hamsters. Animals of G1 (control 1) were killed and the presence of tumors confirmed; G2 (control 2) suffered no interventions for additional 4 weeks; animals of G3 (laser treatment) were irradiated (λ660nm, 50 mW, CW, Ø?=?3 mm, 0.07 cm², 714.2 mW/cm², 133 s, 95 J/cm², 6.65 J) at every other day for 4 weeks; animals of G4 (imiquimod treatment) received 5 % imiquimod three times a week for 4 weeks; and animals of G5 (imiquimod and laser treatment) received both treatments for the same period. Samples were taken and underwent histological analysis by light microscopy and were investigated using immunohistochemistry for S-100⁺ dendritic cells. In G1, G2, and G3, the evaluations showed malignant tumors and the absence of S-100⁺ dendritic cells in the tumor stroma. In G4, 60 % of the animals had no malignant tumors, and S-100⁺ dendritic cells were present in the stroma of the tumors as well as dysplasia. In G5, 40 % of the animals presented SCC, with scarce or no S-100⁺ dendritic cells. The imiquimod treatment played a direct effect on SCC, demonstrated by the increased number of S-100⁺ dendritic cells, which could suggest an important role of immune surveillance against neoplastic proliferation. Furthermore, its association with laser needs to be further investigated.     

Immunological characterization of de novo and recall alloantibody suppression by CTLA4Ig in a mouse model of allosensitization

It is well known that CTLA4Ig inhibits allogenic T-cell activation in transplantation. The immunological features and mechanisms associated with alloantibody suppression by CTLA4Ig, however, are poorly understood. Here, we used a mouse model of allosensitization to evaluate the efficacy of CTLA4Ig (abatacept) in suppression of donor-specific antibody (DSA) during de novo and recall alloantibody responses. We found that abatacept inhibited de novo DSA IgM and IgG responses to HLA-A2 expressing skin grafts. Abatacept administered during primary T cell priming also reduced recall IgG responses induced by re-immunization. Suppression of de novo DSA responses by abatacept is associated with a reduction in splenic expression of the germinal center activation marker GL7 and a reduction of CD4⁺ PD1⁺ CXCR5⁺ follicular T helper (Tfh) subset in splenic lymphocytes detected by flow cytometry. The efficacy of abatacept on recall DSA suppression is moderate. In vitro experiments demonstrated that abatacept inhibited DSA IgG secretion by CD138⁺ plasma cells isolated from allograft recipients. Additional experiments using an IgG1 secreting mouse hybridoma cell line showed that abatacept binds to CD80 expressed on these cells with subsequent inhibition of cell proliferation and reduction in IgG ELISpot formation. In conclusion, CTLA4Ig is a potent suppressor of de novo DSA responses and also affects recall responses. The data suggests modification of recall DSA responses is due to a direct suppressive effect on plasma cells.     

CCL2/MCP‐1 and CXCL12/SDF‐1 blockade by L‐aptamers improve pancreatic islet engraftment and survival in mouse

The blockade of pro‐inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L‐aptamers are chemically synthesized, nonimmunogenic bio‐stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade‐aptamer‐based inhibitors of C‐C Motif Chemokine Ligand 2/monocyte chemoattractant protein‐1 (CCL2/MCP‐1) and C‐X‐C Motif Chemokine Ligand 12/stromal cell‐derived factor‐1 (CXCL12/SDF‐1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2‐specific mNOX‐E36 and the CXCL12‐specific NOX‐A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD‐STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14 days of mNOX‐E36 and NOX‐A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45⁺ leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX‐E36 and NOX‐A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b⁺/Ly6C^high/CCR2⁺ and CD11b⁺/Ly6C^high/CXCR4⁺ cells, respectively. Additionally, both L‐aptamers significantly attenuated diabetes progression in the MLD‐STZ model. In conclusion, CCL2/MCP‐1 and CXCL12/SDF‐1 blockade by L‐aptamers is an efficient strategy to improve islet engraftment and survival.     

Expression of PARP-1 and its active polymer PAR in prostate cancer and benign prostatic hyperplasia in Chinese patients

Background and aims

Aberrant expression of PARP-1 has been reported in various human malignancies and was involved in the progression and metastasis of cancers. However, little is known about PARP-1 expression in prostate cancer (PCa). This study aimed to investigate the expression of PARP-1 and its active polymer poly(ADP-ribose) (PAR) in PCa and benign prostatic hyperplasia (BPH) tissues from Chinese patients.

Methods

The expression of PARP-1 and PAR in PCa and benign prostate hyperplasia tissues was assessed by immunohistochemistry in 78 PCa patients and 49 BPH patients. The relationship between the expression of PARP-1 or PAR and clinicopathological parameters in PCa patients was also analyzed.

Results

Both the positive and strong positive expression rates of PARP-1 in PCa tissues were significantly higher than those in BPH tissues. Although spearman correlations analysis showed the over-expression of PARP-1 and PAR in PCa tissues was not correlated with age, serum PSA level and Gleason scores (GS), an increasing trend was observed between over-expression of PARP-1 or PAR and the PSA levels (TPSA >20 vs TPSA ≤20) or GS grade (GS ≥8 vs GS ≤6).

Conclusion

PARP-1 and PAR expression is markedly elevated in PCa than that in BPH tissues, which may implicate that PARP-1 and PAR are involved in the development of PCa, and the possible expansion in the use of poly(ADP-ribose) polymerase inhibitors for targeting therapy of PCa in select patients alone or combined with chemotherapy or radiation.     

Development of systemic immunologic responses against hepatic metastases during gene therapy for peritoneal carcinomatosis with retroviral HS-tk and ganciclovir

Gene therapy with retroviral mediated gene transfer of the herpes simplex thymidine kinase (HS-tk) gene into a tumor mass confers sensitivity of the tumor cells to ganciclovir (GCV). Tumor-specific immunologic responses may develop following treatment of the primary tumor with retroviral HS-tk and GCV. In the present study we assessed whether GCV treatment of HS-tk transduced colon cancer (TK⁺) implanted in the peritoneal cavity induced a systemic antitumor response that would inhibit growth of a second wild-type (TK⁻) tumor implanted in the live. DHDK12 rat colon cancer cells were transduced in vitro with the retroviral HS-tk vector and established as a permanent cell line (TK⁺ cells). TK⁺ or TK⁻ DHDK12 cells (6×10⁶ cells) were injected intraperitoneally on day 0 into BD-IX rats. On day 10, TK⁻ cells (3×10⁶ cells) were injected into the liver in all the groups. The animals were then treated with GCV (150 mg/kg) for 13 days. TK⁺ peritoneal tumors underwent significant regression during therapy with GCV (0.05±0.004 g; n=7) compared to wild-type (TK⁻) tumors (2.2±0.7 g; n=6) (P<0.05). The volume to TK⁻ tumors in the liver was significantly lower in GCV-treated rats with TK⁺ peritoneal tumors (12.5±8.3 mm³) compared to rats with TK⁻ peritoneal tumors (96.7±18.1 mm³) (P<0.05). Histology of the liver tumors in the TK⁺ groups showed a dense monocytic infiltrate with fibrosis and only occasional viable tumor cells. Gene therapy with retroviral HS-tk vectors may provide a novel approach to treatment of gastrointestinal cancer by both direct cytotoxicity and an indirect mechanism that may included enhanced immunologic responses against disseminated disease. Presented at the Thirty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, San Francisco, Calif., May 19–22, 1996.     

CCL2/CCR2 augments the production of transforming growth factor-beta1, type 1 collagen and CCL2 by human CD45-/collagen 1-positive cells under high glucose concentrations

Background

The migration and activation of circulating profibrotic cells including fibrocytes by the action of the chemokine/chemokine receptor system has been implicated in pathological fibrogenesis. In the present study, the involvement of collagen 1 (Col1)-producing cells, CD45-positive/collagen-1-positive (CD45⁺/Col1⁺) cells originally named as fibrocytes via CC chemokine receptor 2 (CCR2), a cognate receptor of CCL2/monocyte chemoattractant protein, was examined in diabetic conditions.

Methods

Human CD45⁺/Col1⁺ cells originating from the peripheral blood of healthy volunteers were incubated with high concentrations of d-glucose or d-mannitol as an osmotic control for 12, 24 or 48 h. In addition, these cells were preincubated with CCL2 under high glucose concentrations. We also examined the effects of the inhibitors of glucose transporters (GLUTs), reactive oxygen species or CCR2 on the expression of transforming growth factor beta1 (TGF-β₁), pro-α1 chain of Col1 (COL1A1), and CCL2.

Results

Stimulation of CD45⁺/Col1⁺ cells with high glucose concentrations increased the mRNA and protein levels of TGF-β₁ and CCL2 and those of pro-COL1A1, and this effect was mediated in part by increased osmolality. Preincubation of the cells with cytochalasin B (a GLUT inhibitor) or N-acetylcysteine (an antioxidant) blocked the stimulatory effect of high glucose concentrations on these profibrotic molecules. In addition, preincubation of the cells with CCL2 enhanced the high glucose-induced upregulation of TGF-β₁, pro-COL1A1 and CCL2 and migration of the cells, and this effect was partly inhibited by treatment with CCR2 inhibitors.

Conclusion

These results suggest that CD45⁺/Col1⁺ cells may be directly involved, in part through CCL2/CCR2 signaling, in the fibrotic process under diabetic conditions.     

Correlation of the therapeutic effect of activated tumor-draining lymph node cells with specific interferon-γ production in vitro

− CD8⁺ CD25⁺ CD44⁺ T-cells showed specific antitumor efficacy to the pulmonary micrometastases of an autologous tumor, against which lymphokine-activated killer cells were ineffective; however, they did not show cytolytic activity in vitro. The supernatant, obtained by coculturing the activated DLN cells with MC-1 cells, exhibited the specific production of interferon-γ (IFN-γ) which was enhanced by rIL-2. The therapeutic effect of the activated DLN cells correlated with the specific IFN-γ production better than with the cytolytic activity. (Received for publication on July 11, 1997; accepted on May 15, 1998)