Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

Protocol core needle biopsy and histological chronic allograft damage index as surrogate endpoint for Long-Term graft survival.

Following encouraging results from several single-center studies showing that early histological manifestations of chronic rejection are seen in the graft before a decline in transplant function, we tested this concept in a multicenter study and investigated whether protocol needle biopsy may be used as a surrogate to late graft survival in multicenter renal transplantation trials. During two mycophenolate mofetil trials, 621 representative protocol biopsies were obtained at baseline, 1 year, and 3 years. The samples were coded and evaluated blindly by two pathologists and a Chronic Allograft Damage Index (CADI) score was constructed. At 1 year only 20% of patients had elevated (>1.5 mg/100 mL) serum creatinine, whereas 60% of the biopsies demonstrated an elevated (>2.0) CADI score. The mean CADI score at baseline, 1.3 +/- 1.1, increased to 3.3 +/- 1.8 at 1 year and to 4.1 +/- 2.2 at 3 years. The patients at 1 year were divided into 3 groups, those with CADI <2, between 2 and 3.9, and >4.0, the first two groups having normal (1.4 +/- 0.3 and 1.5 +/- 0.6 mg/dL) and the third group pathological (1.9 +/- 0.8 mg/dL) levels of serum creatinine. At 3 years there were no lost grafts in the "low" CADI group, six lost grafts (4.6%) in the "elevated" CADI group, and 17 lost grafts (16.7%) in the "high" CADI group (P <.001). One-year histological CADI score predicts graft survival even when the graft function is still normal. This observation makes it possible to use CADI as a surrogate endpoint in prevention trials and to identify the patients at risk for intervention trials.

Expression of estrogen receptor sub-types alpha and beta in acute and chronic cardiac allograft vasculopathy.

BACKGROUND: The vasculoprotective effects of estrogen are well-established not only in women with age-related atherosclerosis, but also after experimental vascular injury and in chronic allograft vasculopathy. Evidence exists that the newly discovered estrogen receptor (ER) beta, rather than the classical ERalpha is related to the vasculoprotective effect. Here we investigate whether and to what extent the two ERs are expressed in cardiac allografts in the rat and human in native state and during acute and chronic rejection. METHODS: Rat cardiac allografts were performed from male DA (AG-B4, RT1(a)) to male WF (AG-B2, RT1(v)) strain and syngeneic transplants from DA to DA strain; human male-to-male heart allograft endomyocardial biopsies came from our biopsy files. RESULTS: Under in situ hybridization, ERbeta mRNA was prominently expressed in rat vessels and stroma, whereas ERalpha mRNA was present in low levels only. In immunohistochemistry, 2 ERbeta-specific antibodies stained rat and human vessels and stroma, whereas only a weak or no signal was obtained with 2 ERalpha-specific antibodies. Interestingly, the mRNA and protein expression levels in the rat carried only a weak correlation with the intensity of acute rejection, i.e., was not related to the intensity of inflammation. CONCLUSIONS: Our results demonstrate that ERbeta is the predominant ER in rat and human cardiac allografts, and suggest that, unless additional ERs are identified, the vasculoprotective effects of estrogen derivatives in cardiac allograft vasculopathy are mediated by ERbeta rather than by the classical ERalpha.

Estrogen receptor beta dominates in baboon carotid after endothelial denudation injury.

Increasing evidence, mainly from rodents, suggests that the predominant estrogen receptor (ER) in arteries is the newly-described ERbeta. We have investigated the expression of the two ERs in baboon carotid artery before and after denudation injury. Prior to denudation, both full length receptors were detected in semiquantitative RT-PCR; in addition two ERalpha but no ERbeta splicing variants were found. After denudation, ERbeta mRNA increased five-fold and declined, whereas ERalpha mRNA expression remained low. Prior to and after denudation, two ERalpha-specific antibodies showed no reaction with the vessel wall. Instead, two affinity purified antisera to ERbeta demonstrated a weak but distinct reaction over vascular smooth muscle cells with predenudation specimens, escalating post-denudation and declining thereafter. The results suggest that selective targeting to ERbeta should be attempted when designing estrogen-based vasculoprotective drug therapies devoid of uterotrophic side effects.

Structure of carotid artery in baboon and rat and differences in their response to endothelial denudation angioplasty.

BACKGROUND: Most studies dealing with vascular response to injury have been conducted using rodent and rabbit models, although it is expected that the response to injury in these species is dissimilar from man. AIMS: Here we compare the structure of native carotid artery in rat and baboon and the response of these vessels to endothelial denudation angioplasty. METHODS: In both species, the carotid is a musculoelastic artery. Only baboon carotid has a distinct intima, correlating in size with the weight of male baboons. Complete endothelial denudation of left carotid was performed on eight male baboons and 24 male rats by applying an equivalent pull force with a Fogarthy catheter. The animals were sacrificed prior to and 15 min and 2, 3, 4, 7, 14 and 28 days postinjury, one baboon and three rats per time point. RESULTS: Re-endothelialization in the baboon was complete already on day 4, whereas in the rat it was still incomplete on day 28. The proliferative response to injury was far smaller in the baboon than in the rat, the intimal area increased only by 5-fold in baboon compared with 25-fold in rat, and the number of intimal nuclei by 4-fold in baboon compared with 12-fold in rat. Complete compensatory remodelling of the lumen size occurred in the baboon, whereas in the rat remodelling remained incomplete. The cell types participating in the response were, however, similar: deposition of thrombocytes on denuded luminal surface, expression of alpha-actin by intimal cells, and lack of any significant white cell infiltration in the denuded intima. CONCLUSIONS: Baboon carotids are very different from rat carotids both in their native structure and in their response to injury. With the limited amount of information available from human vessels, baboon carotids closely resemble human carotids in both respects.

Role of platelet-derived growth factor in obliterative bronchiolitis (chronic rejection) in the rat.

The role of platelet-derived growth factor (PDGF) in the development of obliterative bronchiolitis (OB) as a manifestation of chronic rejection was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft PDGF-Ralpha and -Rbeta mRNA expression, and in PDGF-AA and -Ralpha immunoreactivity, was demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. Treatment with CGP 53716, a protein-tyrosine kinase inhibitor selective for PDGF receptor, alone and in combination with suboptimal doses of cyclosporin A, significantly reduced myofibroproliferation and the degree of OB by more than 50%. CGP 53716 did not affect airway wall inflammatory cell proliferation, the number of graft-infiltrating CD4(+) or CD8(+) T cells, ED3(+) macrophages, or the level of immune activation determined as IL-2R and MHC class II expression. This study suggests a regulatory role for PDGF, especially for PDGF-AA and -Ralpha, in the development of obliterative bronchiolitis in this model, and demonstrates that inhibition of PDGF receptor protein-tyrosine kinase activation prevents these obliterative changes. Thus, receptor protein-tyrosine kinase inhibitors may provide a novel therapeutic strategy for the prevention of chronic rejection.

High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by alpha(2,3)sialidase(s).

Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors alpha and beta.

Estrogen-based drug therapy in cardiovascular diseases has been difficult because it has not been possible to separate the wanted vasculoprotective effect from the unwanted effects of the hormone to the reproductive system. Here, we demonstrate that, after endothelial denudation of rat carotid artery, the mRNA of the classical estrogen receptor (ERalpha) is constitutively expressed at a low level whereas the expression of the novel ERbeta mRNA increases >40-fold. Under in situ hybridization and immunohistochemistry, ERbeta mRNA and protein colocalize with the smooth muscle cells in the media and neointima. Treatment of ovariectomized female rats with the isoflavone phytoestrogen genistein, which shows 20-fold higher binding affinity to ERbeta than to ERalpha, or with 17beta-estradiol, which does not differentiate between the two receptors, provides similar dose-dependent vasculoprotective effect in rat carotid injury model. In addition in concentrations <10 microM, both ligands are equally inhibitory to the replication and migration of smooth muscle cells in vitro. However, only treatment with 17beta-estradiol, but not with genistein, is accompanied with a dose-dependent uterotrophic effect. The results suggest that preferential targeting to ERbeta will provide vasculoprotective estrogen analogs devoid of effects to the reproductive system.

Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat.

The role of nitric oxide in obliterative bronchiolitis development, i.e., chronic rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in the intragraft inducible nitric oxide synthase (iNOS) mRNA and mononuclear inflammatory cell iNOS immunoreactivity was demonstrated during progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared to syngeneic grafts. In nontreated allografts, however, intragraft nitric oxide production was decreased, most likely because of loss of iNOS epithelial expression. Treatment with aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase, was associated with enhanced proliferation of alpha-smooth muscle actin immunoreactive cells and the intensity of obliterative bronchiolitis early after transplantation. Aminoguanidine treatment did not affect iNOS mRNA synthesis or intragraft nitric oxide production, but decreased iNOS immunoreactivity in smooth muscle cells. Treatment with L-arginine, a precursor of nitric oxide, significantly reduced obliterative changes. L-arginine supplementation enhanced intragraft iNOS mRNA synthesis and iNOS immunoreactivity in capillary endothelial and smooth muscle cells as well as intragraft nitric oxide production. Immunohistochemical analysis of allografts showed that neither iNOS inhibition nor supplementation of the nitric oxide pathway affected the number of graft-infiltrating CD4+ and CD8+ T cells, ED1+ and ED3+ macrophages, immune activation with expression of IL-2R or MHC class II, or production of macrophage or Th1 cytokines. In contrast, L-arginine treatment was associated with increased staining for Th2 cytokines IL-4 and IL-10. In conclusion, this study demonstrates that nitric oxide has a protective role in obliterative bronchiolitis development in this model, and suggests that nitric oxide either directly or indirectly inhibits smooth muscle cell proliferation and modulates immune response towards Th2 cytokines.

Blockade of growth factor synthesis and growth factor action: two possible sites of interference in allograft vessel disease and coronary bypass or balloon injury.

When injured, vascular endothelial cells produce growth factors that cause smooth muscle cells (SMC) to migrate from the media to the intima of the vessel wall, replicate in the intima, and stimulate arteriosclerotic changes. Interference with the actions of growth factors in allograft arteriosclerosis was explored. The somatostatin analog angiopeptin was administered to allograft-recipient rats after transplantation of aortic allografts between major and minor histoincompatible rat strains. Levels of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and platelet-derived growth factor (PDGF) in grafts from angiopeptin-treated recipients were 35% to 75% of levels in grafts from nontreated recipients. Replication of SMC in the media and intima was reduced by 30% to 90% and intimal thickening by approximately 50%. The effect of blockade of IGF-1 receptors (IGF-1R) on the intimal response was also investigated. SMC cultures were serum-deprived of growth factors, then stimulated to replicate by addition of PDGF-B and EGF. Anti-IGF-1 and anti-IGF-1R antibodies reduced SMC replication by 50% and 90%, respectively. A D-amino acid analog of IGF-1, JB3, inhibited SMC replication and dose-dependently inhibited insulin receptor substrate 1 (IRS-1) and IGF-1R phosphorylation in vitro. Infusion of JB3 into rats undergoing balloon dilatation injury inhibited SMC replication in the injured vascular area by nearly 70%, but inhibited intimal thickening by only 30%. In conclusion, interference in the growth factor response may be one way of reducing/preventing vascular injury. However, blockade of more than one growth factor may be needed to achieve an optimal effect.

Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat.

Restenosis after angioplasty is believed to result from stimulation of smooth muscle cells (SMC) by various growth-promoting factors as a consequence of endothelial injury. In this study we have tested the hypothesis that insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) interaction is a rate-limiting step for SMC replication by blocking this interaction with a synthetic D-amino acid peptide structurally resembling the D-domain of IGF-1. After rat carotid artery denudation, semiquantitative PCR analysis demonstrated a significant elevation of IGF-1, platelet-derived growth factor B, transforming growth factor beta 1, and epidermal growth factor mRNAs 10 days after endothelial injury, concomitantly with the induction of intimal SMC proliferation and intimal thickening. Administration of 10-30 micrograms.kg-1.day-1 of D-analog of IGF-1, devoid of proteolytic degradation in body fluids, reduced intimal SMC replication by 60-70%. The peptide also inhibited [3H]TdR incorporation and [3H]glycine incorporation in cultured SMCs by 60-80%, whereas a "scrambled" control peptide consisting of the same amino acids had no effect. The results suggest that IGF-1/IGF-1R interaction is a rate-limiting step for SMC replication. Blocking of this interaction with stabile D-peptide analog of IGF-1 at the level of IGF-1R may offer an entirely new approach for the prophylaxis and treatment of restenosis after cardiac revascularization procedures.