Genetic characterization of Toxoplasma gondii from wild boar (Sus scrofa) in France.

Toxoplasma gondii strains isolated from domestic animals and humans have been classified into three clonal lineages types I-III, with differences in terms of pathogenicity to mice. Much less is known on T. gondii genotypes in wild animals. In this report, genotypes of T. gondii isolated from wild boar (Sus scrofa) in France are described. During the hunting seasons 2002-2008, sera and tissues of individuals from two French regions, one continental and one insular, were tested for Toxoplasma infection. Antibodies to T. gondii were found in 26 (17.6%) of 148 wild boars using the modified agglutination test (MAT, positivity threshold: 1:24). Seroprevalence was 45.9% when considering a threshold of 1:6. Hearts of individuals with a positive agglutination (starting dilution 1:6) (n=60) were bioassayed in mice for isolation of viable T. gondii. In total, 21 isolates of T. gondii were obtained. Genotyping of the isolates using 3 PCR-restriction fragment length polymorphism markers (SAG1, SAG2 and GRA7) and 6 microsatellite loci analysis (TUB2, TgM-A, W35, B17, B18 and M33) revealed that all belonged to type II lineage. These results underline that wild boar may serve as an important reservoir for transmission of T. gondii, and that strains present in wildlife may not be different from strains from the domestic environment.

Lifetime reproductive success and composition of the home range in a large herbivore.

The relationship between individual performance and nonrandom use of habitat is fundamental to ecology; however, empirical tests of this relationship remain limited, especially for higher orders of selection like that of the home range. We quantified the association between lifetime reproductive success (LRS) and variables describing lifetime home ranges during the period of maternal care (spring to autumn) for 77 female roe deer (Capreolus capreolus) at Trois-Fontaines, Champagne-Ardenne, France (1976-2000). We maintained population growth rate (adjusted to account for removals of non-focal animals) near rmax, which enabled us to define the fitness-habitat relationship in the absence of density effects. Using a negative binomial model, we showed that a roe deer's incorporation into its home range of habitat components important to food, cover, and edge (meadows, thickets, and increased density of road allowances) was significantly related to LRS. Further, LRS decreased with increasing age of naturally reclaimed meadows at the time of a deer's birth, which may have reflected a cohort effect related to, but not entirely explained by, a decline in quality of meadows through time. Predictive capacity of the selected model, estimated as the median correlation (rs) between predicted and observed LRS among deer of cross-validation samples, was 0.55. The strength of this relationship suggests that processes like selection of the site of a home range during dispersal may play a more important role in determining fitness of individuals than previously thought. Individual fitness of highly sedentary income breeders with high reproductive output such as roe deer should be more dependent on home range quality during the period of maternal care compared to capital breeders with low reproductive output. Identification of the most important habitat attributes to survival and reproduction at low density (low levels of intraspecific competition) may prove useful for defining habitat value ("intrinsic habitat value").

Design and synthesis of a novel class of histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACs) have emerged as a novel class of antiproliferative agents. Utilizing structure-based design, the synthesis of a series of sulfonamide hydroxamic acids is described. Further optimization of this series by substitution of the terminal aromatic ring yielded HDAC inhibitors with good in vitro and in vivo activities.

New diarylmethylpiperazines as potent and selective nonpeptidic delta opioid receptor agonists with increased In vitro metabolic stability.

Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.

N,N-Diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide: a novel, exceptionally selective, potent delta opioid receptor agonist with oral bioavailability and its analogues.

The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.

Myelointegration of titanium implants: B lymphopoiesis and hemopoietic cell proliferation in mouse bone marrow exposed to titanium implants.

Multinucleated giant cells have been observed at interfaces between bone marrow and titanium implants in mouse femurs. This raises concern that macrophage-derived factors might perturb local lymphohemopoiesis, possibly even predisposing to neoplasia in the B lymphocyte lineage. It has been found that an implant-marrow interface with associated giant cells persists for at least 1.5 years. Precursor B cells show early increases in number and proliferative activity. At later intervals, however, they do not differ significantly from controls, and there are no perturbations in spatial localization of either B lineage cells or DNA-synthesizing hemopoietic cells. The results of this investigation in mice demonstrate that, following initial marrow regeneration and fluctuating precursor B cell activity, and despite the presence of giant cells, titanium implants apparently become well-tolerated by directly apposed bone marrow cells in a lasting state of "myelointegration."

Body mass and individual fitness in female ungulates: bigger is not always better.

In female vertebrates, differences in fitness often correspond to differences in phenotypic quality, suggesting that larger females have greater fitness. Variation in individual fitness can result from variation in life span and/or variation in yearly reproductive success, but no study has yet assessed the relationships between the components of fitness and phenotypic quality while controlling for life span. We tried to fill this gap using data from long-term monitoring (23 years) of marked roe deer and bighorn sheep, two ungulates with very different life histories. In both species, we found a strong positive relationship between an adult female's mass and her probability of reaching old age: over the long term, bigger is indeed better for ungulate females. On the other hand, we found no evidence in either species that heavier females had higher fitness when differences in life span were accounted for: over the short term, bigger is not necessarily better. Our results indicate that, while broad differences in phenotypic quality affect individual fitness, when differences in life span are accounted for phenotypic quality has no residual effect on fitness. Therefore, within a given range of phenotypic quality, bigger is not always better, for reasons which may differ between species.

Substituted (pyridylmethoxy)naphthalenes as potent and orally active 5-lipoxygenase inhibitors; synthesis, biological profile, and pharmacokinetics of L-739,010.

Dioxabicyclooctanyl naphthalenenitriles have been reported as a class of potent and nonredox 5-lipoxygenase (5-LO) inhibitors. These bicyclo derivatives were shown to be metabolically more stable than their tetrahydropyranyl counterparts but were not well orally absorbed. Replacement of the phenyl ring in the naphthalenenitrile 1 by a pyridine ring leads to the potent and orally absorbed inhibitor 3g (L-739,010, 2-cyano-4-(3-furyl)-7-[[6-[3-(3-hydroxy-6,8-dioxabicyclo[3.2.1] octanyl)]-2-pyridyl]methoxy]naphthalene). Compound 3g inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50S of 20, 1.6, and 42 nM, respectively). Derivative 3g is orally active in the rat pleurisy model (inhibition of LTB4, ED50 = 0.3 mg/kg) and in the anesthetized dog model (inhibition of ex vivo whole blood LTB4 and urinary LTE4, ED50 = 0.45 and 0.23 microgram/kg/min, respectively, i.v. infusion). In addition, 3g shows excellent functional activity against ovalbumin-induced dyspnea in rats (60% inhibition at 0.5 mg/kg, 4 h pretreatment) and Ascaris-induced bronchoconstriction in conscious sheep (50% and > 85% inhibition in early and late phases, respectively at 2.5 micrograms/kg/min, i.v. infusion) and, more particularly in the conscious antigen sensitive squirrel monkey model (53% inhibition of the increase in RL and 76% in the decrease of Cdyn, at 0.1 mg/kg, po). In rats and dogs, 3g presents excellent pharmacokinetics (estimated half-lives of 5 and 16 h, respectively) and bioavailabilities (26% and 73% when dosed as its hydrochloride salt at doses of 20 and 10 mg/kg, respectively, in methocel suspension). Based on its overall biological profile, compound 3g has been selected for preclinical animal toxicity studies.

Dioxabicyclooctanyl naphthalenenitriles as nonredox 5-lipoxygenase inhibitors: structure-activity relationship study directed toward the improvement of metabolic stability.

Naphthalenic lignan lactone 3a (L-702,539), a potent and selective 5-lipoxygenase (5-LO) inhibitor, is extensively metabolized at two different sites: the tetrahydropyran and the lactone rings. Early knowledge of the metabolic pathways triggered and directed a structure-activity relationship study aimed toward the improvement of metabolic stability in this series. The best modifications discovered, i.e., replacement of the lactone ring by a nitrile group, replacement of the tetrahydropyran ring by a 6,8-dioxabicyclo[3.2.1]octanyl moiety, and replacement of the pendant phenyl ring by a 3-furyl ring, were incorporated in a single molecule to produce inhibitor 9ac (L-708,780). Compound 9ac inhibits the oxidation of arachidonic acid to 5-hydroperoxy-eicosatetraenoic acid by 5-LO (IC50 = 190 nM) and the formation of leukotriene B4 in human polymorphonuclear leukocytes (IC50 = 3 nM) as well as in human whole blood (IC50 = 150 nM). The good inhibitory profile shown by naphthalenenitrile 9ac is accompanied by an improved resistance to oxidative metabolism. In addition, 9ac is orally active in the functional model of antigen-induced bronchoconstriction in allergic squirrel monkeys (95% inhibition at 0.1 mg/kg).

Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes.

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.

Limited T-cell receptor diversity in liver-infiltrating lymphocytes from patients with primary biliary cirrhosis.

Primary biliary cirrhosis is associated with the presence of high-titer anti-mitochondrial autoantibodies as well as T-cell infiltration of the liver, suggesting the involvement of autoimmune mechanisms. We have studied here the sequences of T-cell receptor alpha and beta chains expressed by T-cell clones derived from liver-infiltrating lymphocytes of two patients with primary biliary cirrhosis. Among the eight clones studied from the first patient, four expressed the same member of the V beta 6 subfamily, associated with either V alpha 4 (three clones) or V alpha 21 (one clone) gene segment. Two other clones expressed an identical V beta 12 transcript, and two in-frame alpha chain transcripts, involving V alpha 2 and V alpha 7 gene segments. From the second patient, eight out of the nine clones were found to rearrange V beta 17-J beta 2.1 and V alpha 3 gene segments. The remaining clone expressed distinct T-cell receptor chains, involving V beta 9 and V alpha 11 gene segments. As deduced from the analysis of their junctional regions, the eight T-cell clones expressing V beta 17/V alpha 3 gene segments derived from only three different T cells. Furthermore, conserved amino acid motifs were found to be encoded in both the alpha and the beta-chain junctional regions. Together, these data show a local amplification of unique T lymphocytes in both patients. The use of identical V beta J beta and V alpha gene segments with similar junctional sequences by three different cells, evidenced in one of the two cases, strengthens the view that liver-infiltrating T lymphocytes are selected locally by autoantigens in PBC.

Substituted thiopyrano[2,3,4-c,d]indoles as potent, selective, and orally active inhibitors of 5-lipoxygenase. Synthesis and biological evaluation of L-691,816.

Thiopyrano[2,3,4-c,d]indoles are a new class of 5-lipoxygenase (5-LO) inhibitors. SAR studies have demonstrated that the thiopyran ring, the 5-phenylpyridine substituent, and an acidic functional group on a four-carbon C-2 side chain are all required for optimal inhibitor potency. In contrast, the indolic nitrogen may be substituted with a variety of lipophilic groups. As a result of the SAR investigation, 44 (L-691,816; 5-[3-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy ]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]-2,2-dimethylpro pyl]-1H- tetrazole) has been identified as a potent inhibitor of the 5-LO reaction both in vitro and in a range of in vivo models. Compound 44 inhibits 5-HPETE production by both rat and human 5-LO and LTB4 synthesis in human PMN leukocytes (IC50s 16, 75, and 10 nM, respectively). The mechanism of inhibition of 5-LO activity by compound 44 appears to involve the formation of a reversible deadend complex with the enzyme and does not involve reduction of the nonheme iron of 5-LO. Compound 44 is highly selective for 5-LO when compared to the inhibition of human FLAP, porcine 12-LO, and also ram seminal vesicle cyclooxygenase. In addition, 44 is orally active in a rat pleurisy model (inhibition of LTB4, ED50 = 1.9 mg/kg; 8 h pretreatment) as well as in the hyperreactive rat model of antigen-induced dyspnea (ED50 = 0.1 mg/kg; 2-h pretreatment). Excellent functional activity was also observed in both the conscious allergic monkey and sheep models of asthma. In the latter case, the functional activity observed correlated with the inhibition of urinary LTE4 excretion.

Effects of cohort, sex, and birth date on body development of roe deer (Capreolus capreolus) fawns.

We investigated the effects of the year of birth (cohort effect), sex and birth date on the variations observed in birth weight and postnatal growth rate in 209 newborn fawns marked during eight consecutive fawning seasons from a highly productive roe deer population under good nutritional conditions. Roe deer exhibited a fast body development with a birth weight of 1628 g and a postnatal growth rate of 139 g/day constant over the first 20 days of their life. As previously reported for dimorphic and polygynous ungulates, there was a marked cohort effect on the rate of body development. This variability was partly explained by climatic conditions during late gestation. When fewer than 5 days with rainfall over 5 mm occurred in April, fawns were heavier. High temperatures during April and during the winter could also be involved in fast body development of roe deer fawns. Sex of fawn did not affect roe deer growth pattern. This was expected on the basis of low sexual dimorphism in size and low polygyny level characteristic of roe deer. Lastly, the date of birth did not affect the body development of roe deer fawns. High constancy of mean birth dates and high synchrony of births observed in this population could account for this result.

Female body size, parity, and malaria infection of Anopheles maculatus (Diptera: Culicidae) in peninsular Malaysia.

The relationship among body size (as indicated by wing length), age (as indicated by parity dissections), and malaria infection were observed in host-seeking Anopheles maculatus Theobald females collected in aboriginal villages of peninsular Malaysia. Both ELISA and salivary gland dissections were used to determine malaria infection. The wings of parous females were significantly longer than those of nulliparous females, suggesting that larger females live longer than smaller ones, and thus have a higher vectorial capacity. Body size differences were not detected between infected parous and uninfected parous females. Females infected with only oocysts were significantly larger than females infected with sporozoites. No correlation was found between the number of oocysts or sporozoites and body size in this small sample.

Effects of interleukin-3 on murine fetal liver hemopoiesis in utero.

The normal regulation of primitive hemopoietic stem cells (PHSCS) throughout development involves locally produced factors and humoral factors. While considerable information is available on the effects of candidate hemopoietic growth factors (HGFs) on postnatal hemopoietic tissues, little is known about the regulatory events of developing hemopoietic stem cells within fetal microenvironments. Fetal hemopoiesis represents expanding populations and may be under different regulatory control mechanisms. The microinjection of purified interleukin-3 (IL-3), a candidate HGF, into 13-day-old mouse fetuses via the yolk sac, allowed us to evaluate its effects on morphogenetic events and, more specifically, on fetal liver populations using quantitative in vitro clonal assays for hemopoietic precursors. In view of the sensitivity of fetal development during the early organogenetic period, considerable care was taken to identify the stress effects of the surgical laparotomy and the microinjection procedure. Control studies, required to distinguish stress effects of surgical laparotomy and microinjection, clearly revealed that the fetal liver is a sensitive organ responding with limited tissue disorganization, reduced cellularity and erythropoietic activity, as monitored 24 h after experimental intervention. The microinjection of 15 units of IL-3 promoted a significant expansion of depleted liver hemopoietic-cell populations and had stimulatory effects on the distribution of connective tissue mast cells and absolute cell numbers, including hemopoietic precursors (erythroid, granulocyte, macrophage, megakaryocyte), compared to controls. These studies suggest that (1) fetal liver hemopoiesis is selectively sensitive to maternal stress but has an effective regenerative capacity to maintain essential hemopoiesis in utero, and (2) fetal hemopoietic cells require an ability to respond to IL-3 early in fetal development.

Further evidence for a gamma/delta T cell receptor-mediated TCT.1/CD48 recognition.

We have demonstrated recently that a molecule, termed TCT.1 (Blast-1/CD48), is recognized on the surface of target cells by a series of alloreactive gamma/delta T cell clones generated from PBL of one healthy individual (designated E). Southern blot analyses suggested that these clones express a TCR associating a V3-JP2-C2 gamma-chain and V1-D-J1-C delta-chain. In the present study, we have developed from PBL of a second normal donor (designated G) a novel series of gamma/delta cloned T cell lines with similar functional activity (i.e., specific recognition of TCT.1 protein). The TCR gamma- and delta-chain nucleotide sequences of both the E and G clones were determined. Results show that 1) sequences from all the clones are identical in each individual donor, 2) the delta-chains expressed by the E and the G clones are encoded by distinct gene rearrangements including V1-D-J delta 1 and V1-D-J delta 2, respectively, 3) the gamma-chains expressed by the E and the G clones are encoded by the same genomic variable elements, namely V gamma 3 and JP2, whereas the junctional regions are distinct. Because the latter rearrangement is very infrequent in human peripheral blood, these data support the view that TCT.1/CD48 recognition is likely to be TCR dependent.

Comparison of urinary leukotriene E4 and 16-carboxytetranordihydro leukotriene E4 excretion in allergic asthmatics after inhaled antigen.

Antisera to 16-carboxytetranordihydro leukotriene E4 (tetranor LTE4), a major urinary oxidative metabolite (via omega- and beta-oxidation) of leukotriene E4 (LTE4) in primates, were obtained by immunisation of rabbits with a related, non-naturally occurring synthetic metabolite (16-carboxytetranordihydro leukotriene C4 ester) conjugated to Keyhole Limpit haemocyanin. Material which competed with [11, 12-3H]tetranor LTE4 for binding to this antisera was isolated from urine from allergic asthmatics by reversed-phase HPLC. This material eluted with the retention time of synthetic standards, and its mean urinary excretion was elevated during both the first three hours (6.13 +/- 2.15 ng/h) and 3-6 h (5.87 +/- 1.99 ng/h) after antigen inhalation, compared with baseline values (3.42 +/- 1.49 ng/h), in 5 allergic mild asthmatics. A much greater and statistically significant increase in urinary leukotriene E4 (LTE4) excretion, occurring in all subjects, was seen during acute antigen-induced bronchoconstriction (baseline, 1.62 +/- 0.66 ng/h; 0-3 h, 19.58 +/- 8.79 ng/h; p less than 0.05) in these subjects. These data support the suggestion that endogenous peptide leukotrienes are metabolised by omega- and subsequent beta-oxidation in man, but emphasize the relative importance of urinary LTE4 excretion after allergen elicited leukotriene generation, further substantiating a pathological role for peptide leukotrienes in allergic asthma.

Metabolism and excretion of exogenous [3H]-LTC4 in primates.

Four novel omega- and beta-oxidation (from the omega end) products of peptide leukotrienes, 20-hydroxy and 20-carboxy-LTE4, 18-carboxy-19, 20-dinor-LTE4 and 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-LTE4 were prepared by total synthesis and used as standards for identification of biliary and urinary metabolites in the cynomolgus monkey. After intravenous administration 14, 15-[3H] leukotriene C4 (10 microCi kg-1) was partially metabolized in and rapidly cleared from the vascular circulation. This resulted, within 24 hours, in significant urinary excretion (14.8 +/- 2.1%, n = 4), consisting largely of material more polar than LTE4 (61% of urinary excretion) as shown by reverse phase HPLC. The polar fraction demonstrated two predominant metabolites which coeluted in several HPLC solvent systems with synthetic 16-carboxytetranordihydro-LTE4 (major component) and 18-carboxydinor-LTE4 (minor component). Characterization of the major polar metabolite as 16-carboxytetranordihydro-LTE4 was substantiated by conversion to its N-acetylated derivative. The absence of the 14, 15 double bond was confirmed by product analysis of oxidative ozonolysis. In a single animal, the bile duct was cannulated, with significant biliary excretion of radioactivity demonstrated over 4 hours (58.6% recovery). The predominant polar biliary metabolites were also identified as the 18-carboxydinor and 16-carboxytetranordihydro derivatives of LTE4 mentioned above. These data suggest that beta-oxidation products generated from the omega-carboxyl end of the 20-carboxy-LTE4 are important products of [3H] LTC4 metabolism in the monkey. Quantitation of these urinary metabolites may be an important index of in vivo leukotriene production.