Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease.

The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

Intra-articular injection of tumor necrosis factor-alpha in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation.

OBJECTIVE: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. DESIGN: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. RESULTS: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. CONCLUSION: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.

Interactions Among a Soil Organic Amendment, Nematodes, and the Nematode-Trapping Fungus Dactylellina candidum.

ABSTRACT When alfalfa leaves (Medicago sativa) are added to soil, both the nematode-trapping fungus Dactylellina candidum and microbivorous nematodes increase. To determine whether the response of the fungus to alfalfa depends on consumption of bacterivorous and fungivorous nematodes, soil microcosm experiments were performed. D. candidum did not increase if alfalfa leaves were added to soil lacking nematodes, but did increase if nematodes were added to soil lacking alfalfa leaves. Although these results indicate that the response of D. candidum to alfalfa depends on nematodes, D. candidum and microbivorous nematodes did not exhibit classical predator-prey dynamics (i.e., D. candidum seldom reduced numbers of resident nematodes and, after initially increasing in alfalfa amended soil, numbers of D. candidum then decreased, whereas numbers of nematodes continued to increase). Fungivorous nematodes were abun dant in alfalfa-amended soil, and their potential to suppress trapping fungi requires more research.

Do Organic Amendments Enhance the Nematode-Trapping Fungi Dactylellina haptotyla and Arthrobotrys oligospora?

Soil cages (polyvinyl chloride pipe with mesh-covered ends) were used to determine how the quantity of two organic amendments affected the nematode-trapping fungi Dactylellina haptotyla and Arthrobotrys oligospora, which were studied independently in two different vineyards. Each cage contained 80 cm(3) of field soil (120 g dry weight equivalent), fungal inoculum (two alginate pellets, each weighing 1.9 mg and containing assimilative hyphae of one fungus), and dried grape or alfalfa leaves (0, 360, or 720 mg equivalent to 0, 4,500, or 9,000 kg/ha) with a C:N of 28:1 and 8:1, respectively. Cages were buried in the vineyards, recovered after 25 to 39 days, and returned to the laboratory where fungus population density and trapping were quantified. Dactylellina haptotyla population density and trapping were most enhanced by the smaller quantity of alfalfa amendment and were not enhanced by the larger quantity of alfalfa amendment. Arthrobotrys oligospora population density was most enhanced by the larger quantity of alfalfa amendment, but A. oligospora trapped few or no nematodes, regardless of amendment. Trapping and population density were correlated for D. haptotyla but not for A. oligospora.

Population Characteristics and Dosage Trajectory Analysis for Mesocriconema xenoplax in California Prunus Orchards.

The Mesocriconema xenoplax population increased exponentially in a newly planted peach orchard. The rate of increase was greater on Nemaguard than on Lovell rootstock and was reduced by postplant nematicides. Population levels were more stable in an established almond orchard on Nemaguard rootstock. All life stages of the nematode were present year round; lower ratios of juveniles to adults in summer suggested adverse effects of temperature and dry soil. Also in summer, there was a smaller proportion of the population in the upper 30 cm of soil than at greater depths. Nematode dosage, average nematode density multiplied by accumulated degree-days (physiological time) of the sampling interval, was useful in quantifying nematode stress on trees and as an indicator of the nematode management effectiveness. The annual trajectory of the nematode dosage could be determined by two samplings, one in spring and one in fall. A nematode predator, the parasitic fungus Hirsutella rhossiliensis, did not regulate ring nematode populations in the newly planted orchard; a recovery period was necessary for increase in the prevalence of parasitism.

Correlations between most probable number and activity of nematode-trapping fungi.

ABSTRACT Soil cages were used to determine whether nematode-trapping fungi population density, as measured by most probable number (MPN) procedures, was correlated with the trapping of nematodes. Fungi studied (and trap type) were Arthrobotrys oligospora (adhesive networks), A. eudermata (adhesive networks), A. dactyloides (constricting rings), Dactylellina ellipsospora (adhesive knobs), and D. haptotyla (adhesive knobs). The fungi were formulated as assimilative hyphae in dried alginate pellets. Pellets were added to field soil, the soil was packed into 80-cm(3) cages (PVC pipe, 3.0 cm long and 3.9 cm in diameter), and the cages were buried in vineyards. After 14 to 61 days, the cages were recovered, and MPN data and trapping activity were determined. For all five fungi, MPN data were correlated with the number of pellets added. Regardless of fungus population density, A. oligospora and A. eudermata trapped few if any nematodes in soil, and consequently, trapping and fungus population density were not correlated. The correlation between population density and trapping was weak for A. dactyloides but relatively strong for D. ellipsospora and D. haptotyla. High levels of trapping by the latter two fungi required more than 10(2) fungus propagules per gram of soil.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Soil pH and the Activity of a Pelletized Nematophagous Fungus.

ABSTRACT When formulated as assimilative hyphae in alginate pellets, the nematophagous fungus Hirsutella rhossiliensis was more active (i.e., parasitized more assay nematodes) in an acidic vineyard soil than in a neutral vineyard soil. To determine whether soil pH explained the difference, fungus activity was measured in soil from the neutral site that had been acidified (by adding sulfuric acid) and in soil from the acidic site that had been neutralized (by adding calcium hydroxide). As hypothesized, the activity of pelletized Hirsutella rhossiliensis was negatively correlated with soil pH. Maximum activity occurred at pH(calcium chloride) 4.5, and activity gradually declined to near zero as the pH increased to 6.5 and rapidly declined to near zero as the pH dropped below 4.0. Assays performed on leached soil samples indicated that the effects of sulfuric acid and calcium hydroxide were largely due to pH rather than to specific ions or osmotic potential. The effect of pH, however, was indirect. Heating the neutral soil to 60 degrees C for 2 h did not alter soil pH or electrical conductivity but increased fungus activity to levels equivalent to those in acidified soil. We conclude that, in these two soils, heat treatment or low soil pH suppresses soil organisms that otherwise interfere with growth of Hirsutella rhossiliensis from alginate pellets.

Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo.

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.

Augmentation of Soil with the Nematophagous Fungi Hirsutella rhossiliensis and Arthrobotrys haptotyla.

In previous studies, growth of Hirsutella rhossiliensis from pelletized assimilative hyphae was reduced by other soil organisms. In the current study, sensitivity to this biotic inhibition was compared when the fungus was added to soil as pelletized hyphae or as fungus-parasitized nematodes. The hypothesis was that the natural inoculum, the parasitized nematode, would be less sensitive than the artificial inoculum, pelletized hyphae. The soil was heated to 60 degrees C for 2 h to remove putative antagonists or was not heated. The soil was packed into vials (17 cm(3)) and kept at 20 degrees C in the laboratory or packed into cages (PVC pipe sealed at the ends with 480-mum pore mesh, 80 cm(3)) and buried 22 cm deep in a vineyard. After 2 or 4 weeks, assay nematodes were added to the vials or cages (recovered from the vineyard), respectively. The assay nematodes were extracted from soil after 2.0 or 2.5 days and examined for adhesive conidia of H. rhossiliensis. Consistent with the hypothesis, H. rhossiliensis was quite sensitive to biotic inhibition when formulated as pelletized hyphae but was insensitive to biotic inhibition when formulated as parasitized nematodes. These data suggest that the activity of the H. rhossiliensis pellet could be increased if the pellet better mimicked the natural inoculum. Similar experiments with the nematode-trapping fungus Arthrobotrys haptotyla, however, exhibited an opposite trend: A. haptotyla was more sensitive to biotic inhibition when added to soil as fungus-parasitized nematodes than as pelletized hyphae. Results from laboratory and field experiments were similar.

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Enchytraeids and nematophagous fungi in tomato fields and vineyards.

ABSTRACT I tested the hypothesis that exclusion of enchytraeids and microarthropods in agricultural fields improves establishment of two nematophagous fungi. Soil was collected from three tomato fields and two vineyards and either heat-treated (2 h at 60 degrees C) or not. Alginate pellets containing hyphae of the fungi Hirsutella rhossiliensis or Monacrosporium gephyropagum were added to the soil, which was packed into cages (PVC pipe, 80-cm(3) volume) sealed with fine (20 mum) or coarse (480 mum) mesh. Cages were buried 22 cm deep in the same fields from which the soil had been collected. After 7 to 50 days, the cages were recovered and fungi and fauna quantified. Fine mesh largely excluded enchytraeids, collembolans, and mites but rarely affected fungus numbers. In contrast, heat treatment of soil rarely affected enchytraeids, collembolans, or mites but frequently increased fungus numbers, regardless of mesh size. The data are inconsistent with the initial hypothesis but are consistent with the idea that organisms narrower than 20 mum interfere with fungal growth from the pellets.

Side effects of brequinar and brequinar analogues, in combination with cyclosporine, in the rat.

Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.

Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy.

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.

Nematode-trapping fungi in organic and conventional cropping systems.

ABSTRACT Nematode-trapping fungi, nematodes, and microbial biomass were quantified in conventionally and organically managed field plots in the Sustainable Agriculture Farming Systems Project at the University of California at Davis. There were four replicate plots (0.135 ha per plot) for each management system, and plots were sampled three times each year for 2 years. The hypothesis that nematode-trapping fungi would be more abundant in organically managed plots was partially supported: the number of species of nematode-trapping fungi was slightly but significantly greater in organic than in conventional plots, two species (Arthrobotrys dactyloides and Nematoctonus leiosporus) were detected more frequently in organic plots, and the population densities of A. dactyloides and N. leiosporus were greater in organic than in conventional plots. Two other species (A. haptotyla and A. thaumasia), however, tended to be more numerous in conventional than in organic plots, and the total density of nematode-trapping fungi was similar in organic and conventional plots. Bacterivorous nematodes were more abundant and microbial biomass (substrate-induced respiration) was greater in organic than in conventional plots. Suppression of the root-knot nematode Meloidogyne javanica, as measured in a bioassay, was not related to management system or population density of nematode-trapping fungi but was positively related to microbial biomass.

Structure-activity relationships (SAR) of some tetracyclic heterocycles related to the immunosuppressive agent Brequinar Sodium.

The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.

Heteroatom- and carbon-linked biphenyl analogs of Brequinar as immunosuppressive agents.

Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.