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.

Successful amplification of DNA specific for Finnish Borrelia burgdorferi isolates in erythema chronicum migrans but not in circumscribed scleroderma lesions.

Early diagnosis of Borrelia burgdorferi infection, hampered by the absence of detectable antibodies in most patients with erythema chronicum migrans is important to prevent late-stage neurologic, rheumatologic, and skin disorders. Furthermore, B. burgdorferi has been claimed to be the causative agent in localized scleroderma (morphea). We used PCR amplification to search for B. burgdorferi outer surface protein OspA-specific sequences in DNA obtained from lesional skin biopsies on Finnish patients with clinically suspect erythema chronicum migrans, lymphocytoma, morphea, or with diverse skin manifestations and persistent high antibodies to B. burgdorferi flagellar antigen. Seronegative patients with other skin lesions served as controls. The amplicons obtained with primers specific for B. burgdorferi type strain B31 ospA sequence did not hybridize to the corresponding probes, and thus the DNA amplified from a Finnish B. burgdorferi erythema chronicum migrans skin isolate was sequenced. This 98-nucleotide sequence of ospA (332-429) showed 11% to 14% nucleotide divergence compared with the North American type strain (B31), several European strains, and an East Siberian tick strain. The sequence was almost identical (99%) to a Swedish isolate from acrodermatitis chronica atrophicans. Using oligonucleotides specific for the Finnish strain, a positive polymerase chain reaction-based hybridization was obtained in six of seven untreated erythema chronicum migrans patients infected in Finland or in Estonia, and in the lymphocytoma patient. Only two of the erythema chronicum migrans patients had IgG or IgM antibodies to flagellin. However, all seven morphea lesions as well as the other lesions were polymerase chain reaction negative. Polymerase chain reaction-based hybridization of B. burgdorferi OspA gene from skin-derived DNA thus provides a sensitive and specific diagnostic tool. In conditions not unequivocally known to be caused by B. burgdorferi, like in morphea, this assay was negative. We also demonstrate that peri-Baltic B. burgdorferi isolates show homology in their OspA genes but differ from geographically more distant isolates.

Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison's disease.

Patients with idiopathic Addison's disease have autoantibodies reacting with adrenal cortex. If Addison's disease is associated with other endocrine immune diseases like autoimmune polyglandular diseases (APD) type I and type II, antibodies may recognize all steroid-producing cells. We showed previously that one antigen recognized by APD-I sera is the cytochrome P450c17 hydroxylase. We have now looked for antibodies to P450c17 and to two other key enzymes in the steroid biosynthetic pathway, the P450scc and P450c21, in a series of patients with isolated Addison's disease (8 patients) or with APD-I or APD-II (50 and 9 patients, respectively). The result of antienzyme antibodies were further correlated with the immunofluorescence pattern against adrenal gland, testis, ovary, and placenta, and with the clinical findings presented. In APD-I patients with Addison's disease and in APD-II patients, antibodies to at least one of the P450 enzymes were frequently found (positive findings in 81% and 78%, respectively). Such antibodies were less frequent in APD-I patients without Addison's disease (21%) and in the isolated Addison cases (25%). In APD-I, antibodies recognized as frequently P450c17 and P450scc, specific for all steroid-producing cells as the adrenal specific enzyme P450c21. In contrast, patients with APD-II or with the isolated Addison's disease reacted almost exclusively with P450c21. Immunofluorescence studies showed good correlation with the known fact that the zona glomerulosa of the adrenal cortex is devoid of the P450c17, that the Leydig cells of the testis and the theca interna cells of the ovary express P450c17 and P450scc, and that the placental trophoblasts express only P450scc. The presence of antibodies to P450scc or to at least one of the tested P450 enzymes correlated significantly to gonadal failure in the females but not in the males.

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).

Effect of mutations in rev gene of SIVmac on virus replication.

The functional activity of SIVmac251 Rev was altered by introducing amino acid changes inside and chain termination mutations after the Rev response element-binding region (RBR) of the protein. The effects of specific mutations were evaluated by transfecting proviral DNAs into the HeLa cell line and into HeLa cells constitutively expressing either HIV-1 Rev or HTLV-1 Rex proteins. Cell-free supernatants from these transient expression assays were further characterized by infecting CD4-positive lymphoid cell lines H9 and MT-4, the latter abortively infected with HTLV-1, and human peripheral blood mononuclear cells. These results together with the data from cotransfection experiments show that SIV can be attenuated up to 95% by introducing changes into the arginine-rich domain, RBR, of Rev. These recessive mutations were efficiently complemented in trans by HIV-1 Rev, SIV Rev, and HTLV-I Rex proteins. In contrast, the mutants of Rev protein that had a chain termination after RBR were trans-dominant negative and could not be trans-complemented with any of these three regulatory proteins. When additional mutations were inserted into the RBR of these trans-dominantly negative Rev proteins, complementation was obtained again.

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.

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.

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.

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.

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.

Identification by molecular cloning of an autoantigen associated with Addison's disease as steroid 17 alpha-hydroxylase.

Idiopathic Addison's disease is characterised by a progressive failure in the synthesis of all classes of steroid hormones and by an immune response against the steroid-producing cells of the adrenal cortex; the nature of the adrenal autoantigens is not known. We have used molecular cloning and sequencing to identify the target antigens. We screened a human fetal adrenal cDNA expression library in lambda gt11 vector with serum samples from patients with Addison's disease as part of the type 1 polyendocrine autoimmunity syndrome. Samples from 3 patients, which had precipitating antibodies against two adrenal proteins detected by immunodiffusion and against five adrenal proteins of molecular mass 55, 48, 43, 39, and 19 kDa as judged by immunoblotting, were used to identify 60 immunoreactive clones. 39 of these were subcloned, inserted into the M13mp10 vector, and sequenced by the dideoxy method or identified by Southern and dot-blot hybridisation. All but 1 of the inserts showed more than 98.8% homology with the published sequence of steroid 17 alpha-hydroxylase. This protein was expressed by insertion of 1 of the clones into the pGEMEX-1 vector. Only serum from patients with Addison's disease and type 1 polyendocrine autoimmunity syndrome that reacted with the 55 kDa adrenal protein recognised the recombinant 17 alpha-hydroxylase protein on immunoblotting. Our results show that one of the key enzymes in steroid biosynthesis, 17 alpha-hydroxylase, is an autoantigen involved in the pathogenesis of adrenocortical failure.

Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines.

Information concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6-9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12-48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.