Screening and activity of potential gastrointestinal probiotic lactic acid bacteria against Yersinia ruckeri O1b.

Yersiniosis of cultured Atlantic salmon is a recurrent fish health management challenge in many continents. The causative organism, Yersinia ruckeri, can reside latently in the gut and lead to acute infection and disease during hatchery and sea-transfer stages. One potential prevention approach is the administration of probiotic bacteria to suppress gut colonization of Y. ruckeri. Our study aimed to isolate and identify anti-Yersinia activity among lactic acid bacteria (LAB) isolated from the gastrointestinal tract (GIT) of aquatic animals. Of the 186 aquatic GIT isolates examined, three strains showed diffusible antimicrobial activity towards Y. ruckeri O1b. Analysis of 16 s rRNA gene sequences indicated the three bacterial strains were Enterococci, related to Enterococcus sp. (99%), Enterococcus thailandicus (99%), and Enterococcus durans (99%). Anti-Yersinia activity was maintained at neutral pH (~6.5-7.0), and in-vitro environmental tolerance assays showed the three strains could withstand simulated salmonids gastrointestinal tract conditions of: low pH (3.4) and 3% bile salt content. All three Enterococci strains showed higher adhesion to the intestinal mucus of Atlantic salmon than Y. ruckeri O1b (E. durans 24%, E. enterococcus sp. 25% and E. thailandicus 98%, compared to Y. ruckeri O1b 5%). However, only Enterococcus sp. and E. thailandicus were able to grow in the salmon intestinal mucus broth while E. durans showed no growth. Anti-Yersinia activity was completely inactivated by proteinase-K treatment, suggesting that the active compound/s are proteinaceous and may be bacteriocin-like inhibitory substances (BLIS). Our data indicate that Enterococcus sp. MA176 and E. thailandicus MA122 are potential probionts for the prevention of yersiniosis in salmonids. Further in-vivo studies are required to determine whether these bacteria reduce the incidence of yersiniosis in Atlantic salmon.

Size-dependent resistance to amoebic gill disease in naïve Atlantic salmon (Salmo salar).

Amoebic gill disease, caused by the protozoan ectoparasite Neoparamoeba perurans, remains a significant threat to commercial Atlantic salmon aquaculture operations worldwide, despite partial control afforded by selective breeding and therapeutic intervention. Anecdotal reports from commercial producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. Here, large (>350 g) and small (<200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L-1 without intervention. Progression and severity of AGD in challenged cohorts was evaluated through gill pathology, using gill score and histological examination, and quantification of gill-associated amoebae burden using qPCR. To determine the potential basis for differences in AGD susceptibility between cohorts, transcriptome analysis was conducted using RNA extracted from whole gill arches. Overall, the large Atlantic salmon cohort had significantly lower gill parasite burdens and reduced AGD-related gross pathology compared to the small cohort. Relative gill load of N. perurans appeared to be proportional to gill score in both size classes, with larger smolts typically observed to have comparatively reduced parasite burdens at a given gill score. Moreover, comparison between gene expression profiles of large and small smolts highlighted upregulation of genes consistent with elevated immune activity in large smolts. Combined, the results presented here provide strong evidence of size-dependent resistance to AGD in AGD-naïve Atlantic salmon.

The Effect of Antimicrobial Treatment upon the Gill Bacteriome of Atlantic Salmon (Salmo salar L.) and Progression of Amoebic Gill Disease (AGD) In Vivo.

Branchial surfaces of finfish species contain a microbial layer rich in commensal bacteria which can provide protection through competitive colonization and production of antimicrobial products. Upon disturbance or compromise, pathogenic microbiota may opportunistically infiltrate this protective barrier and initiate disease. Amoebic gill disease (AGD) is a globally significant health condition affecting salmonid mariculture. The current study examined whether altering the diversity and/or abundance of branchial bacteria could influence the development of experimentally induced AGD. Here, we challenged Atlantic salmon (Salmo salar) with Neoparamoeba perurans in a number of scenarios where the bacterial community on the gill was altered or in a state of instability. Administration of oxytetracycline (in-feed) and chloramine-T (immersion bath) significantly altered the bacterial load and diversity of bacterial taxa upon the gill surface, and shifted the community profile appreciably. AGD severity was marginally higher in fish previously subjected to chloramine-T treatment following 21 days post-challenge. This research suggests that AGD progression and severity was not clearly linked to specific bacterial taxa present in these systems. However, we identified AGD associated taxa including known pathogenic genus (Aliivibrio, Tenacibaculum and Pseudomonas) which increased in abundance as AGD progressed. Elucidation of a potential role for these bacterial taxa in AGD development is warranted.

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

Bacteriomic Profiling of Branchial Lesions Induced by Neoparamoeba perurans Challenge Reveals Commensal Dysbiosis and an Association with Tenacibaculum dicentrarchi in AGD-Affected Atlantic Salmon (Salmo salar L.).

Amoebic gill disease is a parasitic condition that commonly affects marine farmed Atlantic salmon. The causative agent, Neoparamoeba perurans, induces a marked proliferation of the gill mucosa and focal superficial necrosis upon branchial lesions. The effect that amoebic branchialitis has upon gill associated commensal bacteria is unknown. A 16S rRNA sequencing approach was employed to profile changes in bacterial community composition, within amoebic gill disease (AGD)-affected and non-affected gill tissue. The bacterial diversity of biopsies with and without diseased tissue was significantly lower in the AGD-affected fish compared to uninfected fish. Furthermore, within the AGD-affected tissue, lesions appeared to contain a significantly higher abundance of the Flavobacterium, Tenacibaculum dicentrarchi compared to adjunct unaffected tissues. Quantitative PCR specific to both N. perurans and T. dicentrarchi was used to further examine the co-abundance of these known fish pathogens. A moderate positive correlation between these pathogens was observed. Taken together, the present study sheds new light on the complex interaction between the host, parasite and bacterial communities during AGD progression. The role that T. dicentrarchi may play in this complex relationship requires further investigation.

Pigment-depletion in Atlantic salmon (Salmo salar) post-smolt starved at elevated temperature is not influenced by dietary carotenoid type and increasing α-tocopherol level.

Pigment-depletion in the fillets of farmed Atlantic salmon (Salmo salar) arises after periods of elevated water temperatures with voluntary starving. This study tested the effects of dietary pre-loading with different pigment carotenoids (astaxanthin and/or canthaxanthin) combined with two α-tocopherol levels (normal and high: 500 and 1000 mg/kg, respectively) on pigment-depletion in vivo in Atlantic salmon after four weeks of challenge. We also tested whether oxidative stress manifested as an underlying depletion mechanism. Carotenoid levels in whole fillet homogenates were not decreased significantly post-challenge but fillet α-tocopherol concentrations were increased significantly in contrast to decreased oxidative stress indices. However, image analysis revealed localised fillet pigment-depletion following all dietary treatments. These data imply that localised pigment-depletion was not prevented by pre-loading of the fillet with different carotenoid-types/mixtures and increased of α-tocopherol levels from normal to high, respectively. Further, we suggest that oxidative stress might not facilitate pigment-depletion in vivo.

Branchial Pathomorphology of Southern Bluefin Tuna Thunnus maccoyii (Castelnau, 1872) Infected by Helminth and Copepodan Parasites.

Three metazoan parasites, a monogenean Hexostoma thynni and two species of copepods Pseudocycnus appendiculatus and Euryphorus brachypterus are known to parasitize the gills of ranched southern bluefin tuna (SBT) and other tuna species. However, there is no detailed information describing the pathological response to infection by these parasites in this species. Wild southern bluefin tuna Thunnus maccoyii (approximately 3 years of age), captured and towed to a grow-out site in the waters immediately south of Port Lincoln, South Australia were subsequently sampled (n = 10) monthly from March until August 2004 during commercial harvest operations. Longitudinal sections of gill hemibranchs with attached parasites were excised and fixed for routine histology and immunohistochemistry. Reference samples were also collected from fish displaying no signs of parasitism or other grossly observable anomalies. Two morphologically distinct granulocytes were observed and putatively identified as eosinophils and mast cells. Pathology was localized to filaments upon and immediately adjacent to parasite attachment sites. Branchial cellular responses, adjunct to the attachment of H. thynni by its opisthaptoral clamps, included hyperplasia and inflammation resulting in structural remodeling of branchial tissues. Inflammatory infiltrates were often dominated by putative eosinophils and lymphocytes when parasitized by H. thynni and P. appendiculatus. Gill associated lymphoid tissue infiltrated the lamellar regions particularly in response to helminth infection. A variable response ranging from hemorrhage with minor hyperplasia or fibroplasia and eosinophilic inflammation to a barely discernible change was seen for gill sections harboring P. appendiculatus and E. brachypterus. The magnitude of the host response to attachment by the latter was congruent with attachment proximity and parasite load. On the basis of the host responses reported here and the low intensity of infection observed in other associated studies these gill ectoparasites are currently considered a low risk for wild and ranched adult SBT.

Multigenic Delineation of Lower Jaw Deformity in Triploid Atlantic Salmon (Salmo salar L.).

Lower jaw deformity (LJD) is a skeletal anomaly affecting farmed triploid Atlantic salmon (Salmo salar L.) which leads to considerable economic losses for industry and has animal welfare implications. The present study employed transcriptome analysis in parallel with real-time qPCR techniques to characterise for the first time the LJD condition in triploid Atlantic salmon juveniles using two independent sample sets: experimentally-sourced salmon (60 g) and commercially produced salmon (100 g). A total of eleven genes, some detected/identified through the transcriptome analysis (fbn2, gal and gphb5) and others previously determined to be related to skeletal physiology (alp, bmp4, col1a1, col2a1, fgf23, igf1, mmp13, ocn), were tested in the two independent sample sets. Gphb5, a recently discovered hormone, was significantly (P < 0.05) down-regulated in LJD affected fish in both sample sets, suggesting a possible hormonal involvement. In-situ hybridization detected gphb5 expression in oral epithelium, teeth and skin of the lower jaw. Col2a1 showed the same consistent significant (P < 0.05) down-regulation in LJD suggesting a possible cartilaginous impairment as a distinctive feature of the condition. Significant (P < 0.05) differential expression of other genes found in either one or the other sample set highlighted the possible effect of stage of development or condition progression on transcription and showed that anomalous bone development, likely driven by cartilage impairment, is more evident at larger fish sizes. The present study improved our understanding of LJD suggesting that a cartilage impairment likely underlies the condition and col2a1 may be a marker. In addition, the involvement of gphb5 urges further investigation of a hormonal role in LJD and skeletal physiology in general.

Transcriptome profiling the gills of amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.): a role for tumor suppressor p53 in AGD pathogenesis?

Neoparamoeba spp. are amphizoic amoebae with the capacity to colonize the gills of some marine fish, causing AGD. Here, the gill tissue transcriptome response of Atlantic salmon (Salmo salar L.) to AGD is described. Tanks housing Atlantic salmon were inoculated with Neoparamoeba spp. and fish sampled at time points up to 8 days postinoculation (pi.). Gill tissues were taken from AGD-affected fish, and a DNA microarray was used to compare global gene expression against tissues from AGD-unaffected fish. A total of 206 genes, representing 190 unique transcripts, were reproducibly identified as up- or downregulated in response to Neoparamoeba spp. infection. Informative transcripts having GO biological process identifiers were grouped according to function. Although a number of genes were placed into each category, no distinct patterns were observed. One Atlantic salmon cDNA that was upregulated in infected gill relative to noninfected gill at 114 and 189 h pi. showed significant identity with the Xenopus, mouse, and human anterior gradient-2 (AG-2) homologs. Two Atlantic salmon AG-2 mRNA transcripts, designated asAG-2/1 and asAG-2/2, were cloned, sequenced, and shown to be predominantly expressed in the gill, intestine, and brain of a healthy fish. In AGD-affected fish, differential asAG-2 expression was confirmed in samples used for microarray analyses as well as in AGD-affected gill tissue taken from fish in an independent experiment. The asAG-2 upregulation was restricted to AGD lesions relative to unaffected tissue from the same gill arch, while p53 tumor suppressor protein mRNA was concurrently downregulated in AGD lesions. Differential expression of p53-regulated transcripts, proliferating cell nuclear antigen and growth arrest and DNA damage-inducible gene-45beta (GADD45beta) in AGD lesions, suggests a role for p53 in AGD pathogenesis. Thus AGD may represent a novel model for comparative analysis of p53 and p53-regulated pathways.

Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase.

BACKGROUND: Nitration of a critical tyrosine residue in the active site of manganese superoxide dismutase (MnSOD) can lead to enzyme inactivation. In this study, we examined the effect of inducible nitric oxide synthase (iNOS) on MnSOD expression, activity and nitration in acutely rejecting cardiac transplants. METHODS: Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Some rats received L-N6-(1-iminoethyl) lysine (l-NIL), a specific iNOS inhibitor. Protein nitration was determined by immunohistochemical, Western blot and slot-blot analyses. MnSOD enzyme activity and gene expression were determined using Western, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation techniques. RESULTS: MnSOD protein levels were decreased 50% by post-operative day 6 (POD 6), which was prevented by L-NIL. RT-PCR analysis indicated that this decrease could not be explained by any changes in MnSOD mRNA. MnSOD enzyme activity but not protein was decreased at POD 5 in untreated allografts. The loss of MnSOD activity at POD 5 was also prevented by L-NIL. Immunoreactive nitrotyrosine was apparent in untreated allografts at POD 6. Slot-blot analysis indicated that nitrotyrosine formation in allografts could be blocked by L-NIL. Nitration of MnSOD was evident upon immunoprecipitation of MnSOD followed by Western blotting for nitrotyrosine. CONCLUSIONS: These results suggest that the decreased MnSOD enzyme activity in acutely rejecting cardiac allografts can be attributed to a post-translational modification related to nitration arising via an iNOS-dependent pathway. This could be a potential major source of amplified oxidative stress in acute graft rejection.

Hierarchical change in antioxidant enzyme gene expression and activity in acute cardiac rejection: role of inducible nitric oxide synthase.

Reactive oxygen and nitrogen may mediate inflammation injury, but the status of the antioxidant defense system that might influence this process is unknown. In the present study, we examined the expression profile of the antioxidant enzymes, manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GPX) in acutely rejecting cardiac allografts and the potential role of inducible nitric oxide synthase (iNOS) in modulating antioxidant gene expression and activity. Donor hearts from Lewis (isograft) or Wistar-Furth (allograft) rats were transplanted into Lewis recipient rats. A subset of the allografts received L-N6-(1-imino-ethyl) lysine (L-NIL), a specific iNOS inhibitor, beginning the day of surgery until the day of harvesting. Catalase and glutathione peroxidase (GPX) protein levels were significantly decreased by postoperative day 4 (POD4) and postoperative day 5 (POD5), respectively, in allografts compared to isografts. While CuZn superoxide dismutase (CuZn SOD) levels were unchanged, there was a 50% decrease in MnSOD protein in allografts at postoperative day 6 (POD6). The sequential loss in antioxidant protein levels was not due to transcriptional regulation since there was no change in RNA levels for any of the genes tested. L-NIL did not alter catalase protein; however, the loss of MnSOD protein at POD6 was prevented by L-NIL. Consistent with a decrease in antioxidant protein levels, there was a sequential loss in enzyme activity for MnSOD, catalase and GPX. L-NIL however, restored MnSOD and GPX activities but not catalase activity. Treatment with CsA restored both protein and enzyme activities of GPX and MnSOD but not catalase. These results indicate that the loss in MnSOD and GPX protein and activity in allografts occurs via an iNOS-dependent mechanism whereas the decrease in catalase appears to be iNOS-independent. This suggests a differential role for iNOS in regulating post-translational modification of individual antioxidant enzymes in acute cardiac transplantation.

A murine model of invasive aspergillosis: variable benefit of interferon-gamma administration under in vitro and in vivo conditions.

BACKGROUND: Interferon-gamma modulates host defense in a number of infectious diseases. Previous studies have shown that systemic administration of interferon-gamma (IFN-gamma) can enhance survival in experimental invasive aspergillosis (IA). METHODS: Using a novel model of murine IA that is characterized by primary pulmonary infection, we investigated the role of IFN-gamma in the phagocytosis and killing of Aspergillus fumigatus by murine neutrophils and pulmonary alveolar macrophages in vitro and the impact of systemic and regional administration of IFN-gamma on the course of IA in glucocorticoid-treated mice. RESULTS: In vitro, IFN-gamma significantly enhanced phagocytosis and killing function of both neutrophils and alveolar macrophages from normal animals, but not cortisone-treated animals. In vivo, intravenous administration of IFN-gamma did not improve phagocyte recruitment, in vivo killing, or mortality from IA. Regional (intranasal) administration of IFN-gamma to the lungs enhanced recruitment of phagocytic cells to the lungs and improved in vivo killing, but did not alter (and actually worsened) mortality from IA. CONCLUSIONS: The in vitro and in vivo effects of IFN-gamma in IA are contingent on many variables, including the route of administration and the specific pathogenesis of infection.

Human heredity and politics: A comparative institutional study of the Eugenics Record Office at Cold Spring Harbor (United States), the Kaiser Wilhelm Institute for Anthropology, Human Heredity, and Eugenics (Germany), and the Maxim Gorky Medical Genetics Institute (USSR).

Despite the fact that much has been written in recent years about the science of heredity under the Third Reich, there is as yet no satisfying analysis of two central questions: What, if anything, was peculiarly "Nazi" about human genetics under National Socialism? How, under whatever set of causes, did at least some of Germany's most well-known and leading biomedical practioners become engaged in entgrenzte Wissenschaft (science without moral boundaries)? This paper attempts to provide some answers to these two questions comparing three institutes that studied eugenics and human heredity in the 1920s and 1930s: the Eugenics Record Office at Cold Spring Harbor, New York, directed by Charles B. Davenport; the Kaiser Wilhelm Institute for Anthropology, Human Heredity and Eugenics, in Berlin, directed by Eugen Fischer; and the Maxim Gorky Medical Genetics Institute in Moscow, directed by Solomon G. Levit. The institutes are compared on the basis of the kind and quality of their research in eugenics and medical genetics, organizational structure, leadership, patronage (private or state), and the economic-social-political context in which they functioned.

Treatment with {alpha}-phenyl-N-tert-butylnitrone, a free radical-trapping agent, abrogates inflammatory cytokine gene expression during alloimmune activation in rat cardiac allografts.

Spin-trapping nitrones such as alpha-phenyl-N-tert-butylnitrone (PBN) have traditionally been used to trap and stabilize free radicals for detection by electron paramagnetic resonance (EPR) spectroscopy. Unlike classical antioxidants, these agents have never been evaluated therapeutically in allograft transplantation. In the present study, we examined potential mechanisms of action of treatment with PBN in a rat model of acute cardiac allograft transplantation. Graft rejection was determined by histological examination and graft function determined by in situ sonomicrometry. DNA binding for nuclear factor (NF)-kappaB and activator protein (AP-1) were determined by gel shift assays. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for inducible nitric-oxide synthase (iNOS) and inflammatory cytokines. Histological rejection scores were elevated in untreated allografts and decreased by treatment with PBN. In situ sonomicrometry revealed decreased heart rate and distended end diastolic and end systolic segment lengths with rejection. Although PBN did not alter heart rate, it did normalize the distention of both diastolic and systolic cardiac dimension. EPR spectroscopy revealed nitrosylation of myocardial heme protein in untreated allografts that was decreased by treatment with PBN. PBN also decreased iNOS protein and iNOS mRNA. RT-PCR analysis revealed enhanced cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 in untreated allografts. Expression for these genes was potently inhibited or abolished in recipients treated with PBN. PBN treatment also decreased DNA binding of transcription factors, NF-kappaB and AP-1. Thus, PBN retains significant anti-inflammatory properties through its action to down-regulate cytokine gene expression that contribute to protection against acute alloimmune activation in cardiac allografts.

Inhibition of nitrosylation, nitration, lymphocyte proliferation, and gene expression in acute and delayed cardiac allograft rejection by an orally active dithiocarbamate.

Dithiocarbamate derivatives sequester metals such as iron and may have benefits in inflammatory diseases. We examined the actions of a new dithiocarbamate-based oral formulation, NOX-700, on protein modification by nitric oxide (NO), gene expression, and lymphocyte proliferation in a model of acute and delayed cardiac rejection. Chronic treatment with NOX-700 prolonged graft survival. In combination with low-dose cyclosporine (CsA), NOX-700 produced a synergistic action to prolong graft survival. NOX-700 decreased myocardial heme nitrosylation. A single bolus injection with NOX-700 in untreated recipients did not decrease heme nitrosylation but normalized NO metabolites and caused the formation of a mononitrosyl iron complex indicating NO scavenging in vivo. NOX-700 alone given with CsA inhibited protein nitration. NOX-700 or CsA each alone decreased intragraft inflammatory cell infiltration. NOX-700 also potentiated the CsA-induced inhibition of splenocyte proliferation ex vivo stimulated by concanavalin A. In splenocytes derived from treated rats but stimulated ex vivo in a mixed lymphocyte response (MLR), interferon-gamma and cyclin D3 gene expression was inhibited by NOX-700 suggesting down-regulation of lymphocyte activation and proliferation by in vivo treatment. These studies suggest that NOX-700 is protective in cardiac rejection, in part, by scavenging of NO and by limiting lymphocyte activation infiltration.

Variable efficacy of N6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection.

We examined the efficacy and mechanism of action of N(6)-(1-iminoethyl)-L-lysine (L-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. L-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, L-NIL did not alter the increased binding activities for transcription factors nuclear factor-kappaB and activator protein-1. Whereas L-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 microg/ml that incompletely blocked NO production. Higher L-NIL concentrations (30 and 60 microg/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-kappaB binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 microg/ml L-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature.

Beta tryptase levels are not elevated in patients undergoing liver transplantation.

Reperfusion syndrome during liver transplantation and anaphylaxis are clinical syndromes that share similarities in physiological responses. The liver and intestine contain a variety of immunologically active cells, including mast cells. The purpose of this study is to investigate the possibility that mast-cell degranulation occurs routinely during transplantation and thereby contributes to hemodynamic instability and coagulopathy. Beta tryptase, an enzyme released by mast cells during degranulation, is a reliable marker for mast-cell-mediated events. Six patients undergoing liver transplantation had beta tryptase levels assayed in blood immediately before and after reperfusion of the liver. No patient showed an increase in beta tryptase levels at 15 or 60 minutes after reperfusion. In conclusion, it is unlikely that clinically significant mast-cell degranulation occurs routinely in patients undergoing liver transplantation.

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection.

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.

Non-heme iron protein: a potential target of nitric oxide in acute cardiac allograft rejection.

We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis --> Lewis) and mismatched (allografts: Wistar-Furth --> Lewis) rats. On postoperative days (POD) 4-6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl-iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to approximately 50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl-iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl-iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N(6)-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.