Gold(I) ion and the phosphine ligand are necessary for the anti-Toxoplasma gondii activity of auranofin.

Auranofin, an FDA-approved drug for rheumatoid arthritis, has emerged as a promising antiparasitic medication in recent years. The gold(I) ion in auranofin is postulated to be responsible for its antiparasitic activity. Notably, aurothiomalate and aurothioglucose also contain gold(I), and, like auranofin, they were previously used to treat rheumatoid arthritis. Whether they have antiparasitic activity remains to be elucidated. Herein, we demonstrated that auranofin and similar derivatives, but not aurothiomalate and aurothioglucose, inhibited the growth of Toxoplasma gondii in vitro. We found that auranofin affected the T. gondii biological cycle (lytic cycle) by inhibiting T. gondii's invasion and triggering its egress from the host cell. However, auranofin could not prevent parasite replication once T. gondii resided within the host. Auranofin treatment induced apoptosis in T. gondii parasites, as demonstrated by its reduced size and elevated phosphatidylserine externalization (PS). Notably, the gold from auranofin enters the cytoplasm of T. gondii, as demonstrated by scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).IMPORTANCEToxoplasmosis, caused by Toxoplasma gondii, is a devastating disease affecting the brain and the eyes, frequently affecting immunocompromised individuals. Approximately 60 million people in the United States are already infected with T. gondii, representing a population at-risk of developing toxoplasmosis. Recent advances in treating cancer, autoimmune diseases, and organ transplants have contributed to this at-risk population's exponential growth. Paradoxically, treatments for toxoplasmosis have remained the same for more than 60 years, relying on medications well-known for their bone marrow toxicity and allergic reactions. Discovering new therapies is a priority, and repurposing FDA-approved drugs is an alternative approach to speed up drug discovery. Herein, we report the effect of auranofin, an FDA-approved drug, on the biological cycle of T. gondii and how both the phosphine ligand and the gold molecule determine the anti-parasitic activity of auranofin and other gold compounds. Our studies would contribute to the pipeline of candidate anti-T. gondii agents.

Connectomic Basis for Tremor Control in Stereotactic Radiosurgical Thalamotomy.

BACKGROUND AND PURPOSE: Given the increased use of stereotactic radiosurgical thalamotomy and other ablative therapies for tremor, new biomarkers are needed to improve outcomes. Using resting-state fMRI and MR tractography, we hypothesized that a "connectome fingerprint" can predict tremor outcomes and potentially serve as a targeting biomarker for stereotactic radiosurgical thalamotomy. MATERIALS AND METHODS: We evaluated 27 patients who underwent unilateral stereotactic radiosurgical thalamotomy for essential tremor or tremor-predominant Parkinson disease. Percentage postoperative improvement in the contralateral limb Fahn-Tolosa-Marin Clinical Tremor Rating Scale (TRS) was the primary end point. Connectome-style resting-state fMRI and MR tractography were performed before stereotactic radiosurgery. Using the final lesion volume as a seed, "connectivity fingerprints" representing ideal connectivity maps were generated as whole-brain R-maps using a voxelwise nonparametric Spearman correlation. A leave-one-out cross-validation was performed using the generated R-maps. RESULTS: The mean improvement in the contralateral tremor score was 55.1% (SD, 38.9%) at a mean follow-up of 10.0 (SD, 5.0) months. Structural connectivity correlated with contralateral TRS improvement (r = 0.52; P = .006) and explained 27.0% of the variance in outcome. Functional connectivity correlated with contralateral TRS improvement (r = 0.50; P = .008) and explained 25.0% of the variance in outcome. Nodes most correlated with tremor improvement corresponded to areas of known network dysfunction in tremor, including the cerebello-thalamo-cortical pathway and the primary and extrastriate visual cortices. CONCLUSIONS: Stereotactic radiosurgical targets with a distinct connectivity profile predict improvement in tremor after treatment. Such connectomic fingerprints show promise for developing patient-specific biomarkers to guide therapy with stereotactic radiosurgical thalamotomy.

Aztreonam for febrile neutropenia in patients with beta-lactam allergy.

BACKGROUND: Beta-lactam antibiotics are the mainstay of empiric therapy for febrile neutropenia. Aztreonam may benefit certain patients because of a lack of cross-hypersensitivity to penicillins and cephalosporins. This is the first study, to our knowledge, to evaluate the efficacy of aztreonam as monotherapy for febrile neutropenia (FN). METHODS: Our study was a single-center retrospective chart review of patients ≥18 years of age receiving aztreonam for the treatment of FN. Primary outcome was treatment success of aztreonam monotherapy. Secondary analyses included need for modification to antimicrobial therapy, patients transitioned to aztreonam from another empiric regimen, and patients receiving aztreonam in combination with other antibacterial agents. RESULTS: In patients prescribed aztreonam for first fever, 11 of 27 (40.7%) patients who received aztreonam alone and 19 of 40 (47.5%) given aztreonam plus another antibiotic responded within 96 h (P = 0.62). Twenty-four (89%) patients prescribed aztreonam monotherapy were alive when FN resolved or treatment ended. Infectious mortality was low (1 patient, 3.7%). In patients prescribed aztreonam monotherapy following an adverse reaction to cefepime, 6 of 11 (54.5%) responded within 96 h of initiating aztreonam; 10 (91%) were alive when FN resolved or treatment ended. CONCLUSION: Aztreonam monotherapy may be acceptable for use in patients with a history of beta-lactam hypersensitivity or following an adverse reaction with another beta-lactam. Further studies are needed to compare efficacy of aztreonam monotherapy with other therapies for the treatment of FN.

Apo(a) promotes thrombosis in a vascular injury model by a mechanism independent of plasminogen.

OBJECTIVE: Structural similarity between apolipoprotein(a) [apo(a)], the unique apoprotein of lipoprotein(a), and plasminogen (Plg), the zymogen for plasmin, results in inhibition of functions of Plg by apo(a) in vitro. The objective of this study was to evaluate the interaction of Plg and apo(a) in vivo. METHODS AND RESULTS: Vascular injury was induced in the carotid artery with a perivascular cuff in: (i) wild-type (WT); (ii) Plg deficient (Plg-/-); (iii) apo(a) (6 KIV construct) transgenic [apo(a)tg]; and (iv) apo(a) transgenic and Plg deficient [apo(a):Plg-/-] mice. At 10 days after cuff placement, the media and adventitia area were increased in the injured carotids compared with the uninjured carotids, and collagen deposition was greater in apo(a)tg, Plg-/- and apo(a):Plg-/- mice compared with WT mice. The incidence of a thrombus was greater (P < 0.05) in apo(a):Plg-/- mice (83%) than WT (20%), Plg-/- (12%), and apo(a)tg mice (9%). In the thrombi from apo(a)tg and apo(a):Plg-/- mice, P-selectin and von Willebrand factor immunostaining, indicating a platelet-rich thrombi, was greater than in WT and Plg-/- mice. The presence of fibrin(ogen) in the thrombi was greater in Plg-/- and apo(a):Plg-/- mice than apo(a)tg and WT mice. Of the four genotypes, only the apo(a):Plg-/- mice had both increased platelet and increased fibrin(ogen) deposition. CONCLUSIONS: The major finding of this study is the high incidence of thrombosis after vascular injury in apo(a)transgenic mice in a Plg deficient background, providing strong evidence for a prothrombotic role of apo(a) independent of Plg in vivo.

Complement activation in postpartum thyroiditis.

BACKGROUND: Postpartum thyroid dysfunction (PPTD) develops in 50% of pregnant women who have raised levels of circulating thyroid peroxidase autoantibodies (TPOAb) at booking. Although these antibodies are able to activate the complement cascade in vitro, it is not known whether complement activation plays any role in the pathogenesis of this disease. AIM: To investigate potential and actual activation of the complement system in women with postpartum thyroiditis. DESIGN: Complement activation was monitored on a weekly basis in 24 postpartum women who had raised TPOAb at 16 weeks gestation, attending an antenatal clinic in Mid-Glamorgan, Wales. METHODS: ELISA procedures were used to measure both in-vitro complement C3 activation by TPOAb and circulating terminal complement complexes (TCC) in serum. RESULTS: Higher levels of bioactive TPOAb activity were seen in women who developed PPTD when compared to those who did not. However, TCC remained undetectable in serum throughout the period of study. CONCLUSIONS: In PPTD, despite the presence of circulating bioactive TPOAbs, the extent of complement activation is inadequate to cause detectable increases in peripheral blood TCC, suggesting that the complement system may not play a major role in PPTD pathogenesis.

Tg.AC genetically altered mouse: assay working group overview of available data.

In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

CB6F1-rasH2 mouse: overview of available data.

This article presents data from short-term carcinogenicity studies of compounds tested in the CB6F1-rasH2 transgenic mouse as part of the International Life Sciences Institutes' (ILSI) Health and Environmental Sciences' (HESI) Alternative to Carcinogenicity Testing (ACT) project. Additionally, data from other studies that were not conducted as part of the ILSI program, but used comparable or slightly modified protocols, are included here. A significant number (3 of 4) of the genotoxic carcinogens tested were positive in the rasH2 mouse; the other compound was equivocally positive. The positive control, N-Methyl-N-nitrosurea (MNU), gave reproducible responses across all participating laboratories with tumors noted at multiple sites in the animal. The immunosuppressive human carcinogen. Cyclosporin A, was equivocal. Two hormones that are human tumorigens. Diethylstilbestrol and 17beta-Estradiol, gave positive and negative results, respectively. Of the twelve additional compounds tested that are classified as non-genotoxic rodent carcinogens and putative human non-carcinogens, only the two peroxisome proliferators (clofibrate and diethylhexylphthalate(DEHP)) produced a positive response (liver effects). The three non-genotoxic non-carcinogens that were tested also gave negative responses in the rasH2 model. This result provides confidence that the model is likely to have a low false-positive rate.

Static magnetic field therapy for pain in the abdomen and genitals.

Two adolescents with debilitating, medication-resistant, chronic pain of the low back and abdomen with intermittent pain of the genitalia were diagnosed with intervertebral disk disease at spinal cord levels that correlated with their signs. Both patients had undergone multiple evaluations by physicians of different specialties and both underwent appendectomy without relief of their pain. The history of the onset of pain was important in determining the affected levels. The pain of both individuals was mimicked and localized by percussion of the vertebral spines at the level of disk protrusion. This maneuver and careful review of the history were important in making the correct diagnosis in each case. In both patients, treatment with novel magnetic devices provided rapid relief that was sustained for more than 2 years. These cases highlight the need for careful evaluation and correct diagnosis of abdominal and genital pain in young patients to avoid costly and unnecessary medical intervention and the stigma of painful debility.

Genotype spectrum of ornithine transcarbamylase deficiency: correlation with the clinical and biochemical phenotype.

Ornithine transcarbamylase (OTC) deficiency, a partially dominant X-linked disorder, is the most common inherited defect of the urea cycle. Previous reports suggested a variable phenotypic spectrum, and several studies documented different "private" mutations in the OTC genes of patients. Our laboratory identified disease-causing mutations in 157 families with OTC deficiency, 100 of which came to medical attention through a hemizygous propositus and in 57 the index case was a heterozygous female. We correlated the genotype with age of onset, liver OTC activity, incorporation of nitrogen into urea, and peak plasma ammonia levels. The "neonatal onset" group has a homogeneous clinical and biochemical phenotype, whereas the "late onset" group shows an extremely wide phenotype; 60% of the mutations are associated exclusively with acute neonatal hyperammonemic coma. The remaining mutations caused a nonuniform phenotype ranging from severe disease to no symptoms; 31% of the mutations in the OTC gene occur in CpG dinucleotides (methylation-mediated deamination), and none of them accounted for more than 4% of the total. Eighty-six percent of the mutations represented single-base substitutions and 68% of the substitutions were transitions. G-to-A and C-to-T transitions were the most frequent substitutions (34 and 21%, respectively) whereas C-to-A, A-to-C, C-to-G, and T-to-A transversions were the least common (1.5-3%). Twenty percent of propositi and 77% of propositae carried new mutations. Forty percent of female germinal mutations were in CpG dinucleotides whereas this number appears much smaller in male germinal mutations. These data allow classification of patients with OTC deficiency into at least two groups who have discordant disease course and prognoses. In addition, they improve our understanding on the origin of mutations in the OTC gene and allow better counseling of affected families.

Antibody cross-linking of human CD9 and the high-affinity immunoglobulin E receptor stimulates secretion from transfected rat basophilic leukaemia cells.

Previous studies have shown that antibody cross-linking of the tetraspanin protein CD9 stimulates the degranulation of platelets and eosinophils, although the mechanism of activation is unclear. In this work we transfected human CD9 into the rat basophilic leukaemia (RBL-2H3) cell line and studied the stimulation of secretion from these cells in response to a panel of anti-CD9 antibodies. Intact immunoglobulin G1 (IgG1) antibodies activated transfected cells whereas F(ab')2 fragments of antibody and an intact IgG2a did not. Stimulation of secretion was inhibited by co-incubation with monomer murine immunoglobulin E (IgE) but not with an IgG1 isotype control, indicating that the response involves the endogenous high-affinity IgE receptor (FcepsilonRI). The anti-CD9 antibody activation curve was biphasic, and supraoptimal antibody concentrations stimulated little or no degranulation, indicating that multivalent binding of human CD9 molecules is necessary for the formation of an active complex with rat FcepsilonRI. Immunoprecipitation of FcepsilonRI under mild detergent conditions co-precipitated CD9, suggesting the presence of pre-existing complexes of CD9 and FcepsilonRI that could be activated by antibody cross-linking. These data are further evidence that tetraspanins are involved in FcepsilonRI signalling and may reflect the participation of tetraspanins in the formation of complexes with other membrane proteins that use components of Fc receptors for signal transduction.

Platelet kinetics in dogs treated with a glycoprotein IIb/IIIa peptide antagonist.

Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists have been highly effective inhibitors of platelet aggregation in preclinical studies and in clinical trials. However, decreased platelet counts have been documented in preclinical studies and in some patients receiving GPIIb/IIIa antagonists. We evaluated changes in platelet kinetics and fate in dogs receiving the GPIIb/IIIa receptor antagonist RPR 109891 orally for 4 days. Dogs receiving RPR 109891 had a 22-52% decrease in platelet count with the nadirs at 3-5 days after initiation of treatment. Platelet survival time was reduced by 19%, and platelet half-life was reduced by 63%. Indium-111-labeled platelets were rapidly cleared from the blood within 1 hour after administration of RPR 109891 on treatment days 1 and 2. This clearing was associated with a sharp increase in radioactivity in spleen but not in liver or lung. Platelet clearance was markedly attenuated on treatment days 3 and 4. Platelet counts returned to baseline within 1 week after discontinuation of treatment. These data indicate that RPR 109891 causes rapid and selective sequestration of platelets in the spleen.

The molecular basis of ornithine transcarbamylase deficiency.

UNLABELLED: The ornithine transcarbamylase (OTC) gene is located on the short arm of the X-chromosome and encodes the second enzyme of the urea cycle. OTC deficiency is an X-linked disorder that causes hyperammonemia leading to brain damage, mental retardation and death. The clinical and biochemical phenotype is extremely variable and can only partially be explained by the genotype. We identified mutations in the OTC gene of more than 150 patients with OTC deficiency. The "neonatal onset" group of patients has mutations that abolish enzyme activity, whereas the "late onset group" shows partial enzyme deficiency to variable degree. Of the mutations, 60% are associated exclusively with acute neonatal hyperammonemic coma while the remaining cause "late onset" disease. Several symptomatic and asymptomatic adults have now been identified to have deleterious mutations in the OTC gene leading to predisposition to hyperammonemia. CONCLUSION: The enlarging clinical, biochemical and molecular spectrum observed in patients with ornithine transcarbamylase deficiency suggests that this disorder behaves like a single gene disorder at one end of the spectrum and as a multi-factorial disease at the other.

Gene therapy vectors based on adeno-associated virus type 1.

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.

TCDD residues in fish and shellfish from U.S. waterways.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) in the edible portion of fish and shellfish from various U.S. waterways has been monitored since 1979. Analytical results for the period 1979-1994 are reported. Extracts obtained after column chromatographic and liquid chromatographic cleanup were examined by electron capture detection-gas chromatography (GC), and final quantitation and confirmation were performed by GC/mass spectrometry with multiple ion detection. Analyses of 1623 test samples indicated that TCDD residues in fish and shellfish were not widespread but rather were localized in areas near waste sites, chlorophenol manufacturers, and pulp and paper mills. Analytical results indicated that levels in aquatic species from these sites have been declining steadily. No TCDD (limit of detection and confirmation, 1-2 ppt) has been found in recent years in aquatic species from most Atlantic, Pacific, and Gulf of Mexico sites and Great Lakes other than Lake Ontario and Saginaw Bay (Lake Huron).

In vitro dissolution profile of water-insoluble drug dosage forms in the presence of surfactants.

The determination of the in vitro release profile of water-insoluble drug products requires dissolution media different from those used for water-soluble drug products. Since the relevance of drug dissolution in organic solvents is questionable, we investigated the use of surfactants to determine the dissolution profiles of water-insoluble drug products. In most cases, the drug dissolution rate and extent increased as the surfactant concentration in the aqueous dissolution medium increased. Suitable dissolution profiles were obtained in the presence of sodium lauryl sulfate (SLS) for water-insoluble drug products, such as griseofulvin, carbamazepine, clofibrate, medroxyprogesterone, and cortisone acetate. These findings recommend the use of surfactants for determining the aqueous dissolution of water-insoluble drug products rather than adding organic solvents to the dissolution medium.

The role of visual similarity in picture categorization.

Categorization is usually assumed to require access to a concept's meaning. When pictures are categorized faster than words, they are assumed to be understood faster than words. However, pictures from the same category are more similar than pictures from different categories. The present article argues that the use of visual similarity as a cue to category membership may produce the picture advantage. The visual similarity hypothesis was tested in two experiments. In the first experiment, pictures showed a disadvantage for the visually similar categories of fruits and vegetables, but showed their usual advantage for the visually dissimilar categories of fruits and animals. In the second experiment, with a mixed list design, pictures were slower only for visually similar different decisions, but showed the usual advantage for all other decisions. The reliability of visual similarity as a cue to the decision accounted well for these results. Because visual similarity can be shown to have large effects on picture categorization, the use of categorization to compare speed of understanding of pictures and words is questionable.

Pulmonary fibrosis with small-airway disease: a model in nonhuman primates.

Bleomycin was administered intrabronchially to four baboons in doses of 1 mg/kg for four consecutive weeks. At necropsy 6 months later, the lesions produced differed markedly from those resulting from parenteral administration of bleomycin and consisted of diffuse foci of inflammation and fibrosis of the lung parenchyma associated with small airway lesions. Airway lesions were found in respiratory bronchioles and consisted of bronchiolar wall inflammation, hyperplasia of smooth muscles, and epithelia bronchiolization of adjacent alveolated structures. Many bronchioles were obliterated by the fibrotic process. Biochemical measurements confirmed the histologic appearance of increased lung collagen in three of four animals. These findings indicate that obstruction of small airways by processes which cause lung fibrosis may be separable physiologically from processes which affect only the lung parenchyma.