Synthesis of a Reactive Oxygen Species-Responsive Doxorubicin Derivative.

A heterobifunctional reactive oxygen species (ROS)-responsive linker for directed drug assembly onto and delivery from a quantum dot (QD) nanoparticle carrier was synthesized and coupled to doxorubicin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/sulfo⁻NHS coupling. The doxorubicin conjugate was characterized using ¹H NMR and LC-MS and subsequently reacted under conditions of ROS formation (Cu2+/H2O2) resulting in successful and rapid thioacetal oxidative cleavage, which was monitored using ¹H NMR.

A pathogenic role for tumor necrosis factor-related apoptosis-inducing ligand in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory respiratory disorder, often induced by cigarette smoke (CS) exposure. The development of effective therapies is impaired by a lack of understanding of the underlining mechanisms. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with inflammatory and apoptotic properties. We interrogated a mouse model of CS-induced experimental COPD and human tissues to identify a novel role for TRAIL in COPD pathogenesis. CS exposure of wild-type mice increased TRAIL and its receptor messenger RNA (mRNA) expression and protein levels, as well as the number of TRAIL(+)CD11b(+) monocytes in the lung. TRAIL and its receptor mRNA were also increased in human COPD. CS-exposed TRAIL-deficient mice had decreased pulmonary inflammation, pro-inflammatory mediators, emphysema-like alveolar enlargement, and improved lung function. TRAIL-deficient mice also developed spontaneous small airway changes with increased epithelial cell thickness and collagen deposition, independent of CS exposure. Importantly, therapeutic neutralization of TRAIL, after the establishment of early-stage experimental COPD, reduced pulmonary inflammation, emphysema-like alveolar enlargement, and small airway changes. These data provide further evidence for TRAIL being a pivotal inflammatory factor in respiratory diseases, and the first preclinical evidence to suggest that therapeutic agents that target TRAIL may be effective in COPD therapy.

Alpha and gamma herpesvirus detection in two herds of farmed red deer (Cervus elaphus) in New Zealand.

CASE HISTORY: In September 2004 two hinds on Farm 1 were observed with epiphora and keratoconjunctivitis, and corneal scarring. A low pregnancy rate in some hinds had been recorded that year. In the same year six yearling deer were observed on Farm 2 with keratitis, uveitis and corneal scarring. CLINICAL AND PATHOLOGICAL FINDINGS: On Farm 1, conjunctival swabs and blood samples were collected from the hinds with ocular lesions, and from 24 other hinds. The two affected hinds were immunosuppressed with dexamethasone for 7 days. Conjunctival, nasal and vaginal swabs were collected daily before euthanasia and necropsy on the eighth day. Subsequently, another five non-pregnant hinds were similarly immunosuppressed and necropsied, and the reproductive tracts of 20 non-pregnant hinds were collected following slaughter. Semen samples were collected from four stags implicated with reproductive failure. On Farm 2, conjunctival swabs were collected from six hinds with ocular lesions and from 14 unaffected deer. Viral culture, consensus primer PCR and sequencing for specific herpesviruses was carried out on conjunctival swabs, buffy coat from blood samples, semen and reproductive tracts. Necropsy samples were also examined using gross pathology and histopathology. On Farm 1, a type 2 rhadinovirus (CvRhV) was detected in the conjunctiva of one hind with keratoconjunctivitis using PCR. Following immunosuppression, gross vesicular and histological vaginal lesions typical of infection with alphaherpesvirus were observed in samples of vaginal tissue from the same hind. Buffy coat, vaginal and lumbar spinal nervous tissues were also positive for cervid herpesvirus 1 (CvHV-1) using PCR. Herpesviruses were not detected in reproductive tracts, ocular or semen samples of the other deer. CvRhV was detected in buffy coats from four other hinds and in a conjunctival swab from one hind, all without ocular lesions, using PCR. On Farm 2, conjunctival swabs from two deer with keratitis were culture positive for CvHV-1. Two culture-negative conjunctival samples from deer without ocular lesions were positive for CvHV-1 by PCR. In two other affected animals, presence of CvRhV was confirmed by PCR and sequencing. DIAGNOSIS: Infection with CvHV-1 associated with keratitis and vulvovaginitis, and CvRhV infection in deer with and without ocular lesions. CLINICAL RELEVANCE: CvHV-1 is a likely cause of keratoconjunctivitis and possibly reproductive tract pathology in deer. Investigation of ocular lesions and reproductive failure in farmed deer should include CvRhV and CvHV-1.

Poly[aqua(µ-vinyl-phospho-nato)cadmium].

The title compound, [Cd(C(2)H(3)O(3)P)(H(2)O)](n), was obtained from vinyl-phospho-nic acid and cadmium nitrate. The vinyl groups project into the inter-lamellar space and the structure is held together via van der Waals forces. The Cd(2+) ion is six-coordinate and the geometry is best described as distorted octa-hedral, with O-Cd-O angles falling within the range 61.72 (13)-101.82 (14)°. Five of the coordinated oxygen atoms originate from the phospho-nate group and the sixth from a bound water molecule. Cd-O distances lie between 2.220 (3) and 2.394 (2) Å. The water mol-ecule is hydrogen bonded to a phospho-nate oxygen atom.

Differential effects of Co(III), Ni(II), and Ru(III) amine complexes on Sindbis virus.

Transition metal complexes [Co(cyclen)(NH(3))(2)](ClO(4))(3)H(2)O (cyclen=1,4,7,10-tetraazacyclododecane) (2), [Co(NH(3))(5)(OH(2))](CF(3)SO(3))(3) (3) [Ni(NH(3))(6)]Br(2) (4) and [Ru(NH(3))(6)]Cl(3) (5) were tested against Sindbis infected baby hamster kidney (BHK) cells and show differential effects from the previously reported anti-viral complex [Co(NH(3))(6)]Cl(3) (1). The macrocyclic complex 2 and labile aqua complex 3 show either no or little effect on the survival on Sindbis virus-infected cells as compared to that for 1, which show a monotonic increase in % BHK cell survival. Nickel and ruthenium ammine complexes 4 and 5 had a moderate influence of cell survival. While the results showed some anti-viral activity for some of the structural variations, it appears that 1, with its potential to be a broad-spectrum anti-viral compound, occupies a unique position in its ability to both significantly enhance cell survival and to decrease viral expression of infected cells. We also show that 1 also shows anti-viral activity against Adenovirus lending support to the broad-spectrum potential of this complex.

Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells.

The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects. Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy atopic nonasthmatic or atopic asthmatic children. Gene expression stability was determined using three different Visual Basic for Applications applets (geNorm, NormFinder and BestKeeper). All 12 HKGs were expressed in AECs. However, the hypoxanthine ribosyltransferase and TATA-binding protein genes were excluded from further analysis due to low expression levels. The cyclophilin A gene was ranked the most stable by all three methods. The expression levels of the beta-actin and glyceraldehyde-3-phosphate dehydrogenase genes were significantly different between the three groups of patients, with atopic asthmatics showing the highest expression levels for both genes. The results suggest that the cyclophilin A gene is the most suitable housekeeping gene analysed for expression studies utilising uncultured bronchial airway epithelial cells from healthy and asthmatic children, and highlight the importance of validating housekeeping genes for each experimental model.

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays.

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.

Benign solitary schwannomas: a review of 234 cases.

We reviewed 234 benign solitary schwannomas treated between 1984 and 2004. The mean age of the patients was 45.2 years (11 to 82). There were 170 tumours (73%) in the upper limb, of which 94 (40%) arose from the brachial plexus or other nerves within the posterior triangle of the neck. Six (2.6%) were located within muscle or bone. Four patients (1.7%) presented with tetraparesis due to an intraspinal extension. There were 198 primary referrals (19 of whom had a needle biopsy in the referring unit) and in these patients the tumour was excised. After having surgery or an open biopsy at another hospital, a further 36 patients were seen because of increased neurological deficit, pain or incomplete excision. In these, a nerve repair was performed in 18 and treatment for pain or paralysis was offered to another 14. A tender mass was found in 194 (98%) of the primary referrals. A Tinel-like sign was recorded in 155 (81%). Persistent spontaneous pain occurred in 60 (31%) of the 194 with tender mass, impairment of cutaneous sensibility in 39 (20%), and muscle weakness in 24 (12%). After apparently adequate excision, two tumours recurred. No case of malignant transformation was seen.

Severe sequelae after stonefish envenomation.

More tourists are visiting the idyllic waters of the South Pacific each year. They share the shallow waters with the native wildlife, including the venomous stonefish. Although the injection of its venom has been reported to have occasional fatal neuromuscular and cardiovascular sequelae, severe tissue necrosis at the injection site has not been described. We present a case of stonefish injury that describes serious localised sequelae of stonefish envenomation

Inducible NO synthase expression is low in airway epithelium from young children with cystic fibrosis.

BACKGROUND: This is the first study to measure inducible nitric oxide synthase (iNOS) gene and protein expression quantitatively in primary epithelial cells from very young children with cystic fibrosis (CF). Low levels of exhaled nitric oxide (NO) in CF suggest dysregulation of NO production in the airway. Due to the importance of NO in cell homeostasis and innate immunity, any defect in the pathway associated with CF would be a potential target for treatment. METHODS: Cells were obtained by tracheobronchial brushing from 40 children with CF of mean (SD) age 2.1 (1.5) years and from 12 healthy non-atopic children aged 3.4 (1.2) years. Expression of iNOS mRNA was measured using quantitative PCR and iNOS protein by immunofluorescence and Western blot analysis. RESULTS: Inducible NOS mRNA expression was significantly lower in CF patients with and without bacterial infection than in healthy children (0.22 and 0.23 v 0.76; p=0.002 and p=0.01, respectively). Low levels of iNOS gene expression were accompanied by low levels of iNOS protein expression as detected by Western blot analysis. CONCLUSIONS: These results support the findings of previous studies in adult patients with advanced disease, cell lines, and animal models. Our findings reflect the situation in children with mild lung disease. They indicate that low iNOS expression may be an innate defect in CF with potential consequences for local antimicrobial defence and epithelial cell function and provide evidence for an approach to treatment based on increasing epithelial NO production or the sensitivity of NO dependent cellular processes.

Peri-acetabular radiolucent lines: inter- and intra-observer agreement on post-operative radiographs.

Peri-acetabular radiolucent lines (RLLs) seen on "early" post-operative radiographs have been identified as a potential predictor of long-term implant performance. This study examines the inter- and intra-observer variation encountered when assessing such radiographs. Four consultant orthopaedic surgeons assessed the presence, extent and width of RLLs in 220 radiographs performed on 50 patients taken one to two weeks, six weeks, six months and one year following surgery. Inter-observer agreement was fair at 7-14 days but improved to moderate to good in films at six and 12 months. Intra-observer agreement was moderate to good at 7-10 days but again improved to good at 6 and 12 months. When only the presence or absence of RLLs was considered, both inter-observer and intra-observer agreement improved for both the six-month and one-year radiographs. This experiment shows that caution must be used for the interpretation of RLLs on hip radiographs taken during the very early post-operative period. We recommend that films taken at least six weeks to six months following surgery should be used for assessment to reduce observer variation. For optimum results, a single experienced observer should do the assessment with a simple classification.

Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct.

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath.

BACKGROUND: The fractional concentration of nitric oxide (NO) in exhaled breath (FeNO) is increased in asthma. There is a general assumption that NO synthase (NOS) 2 in epithelium is the main source of NO in exhaled breath. However, there is no direct evidence to support the assumption and data from animal models suggest that non-inducible NOS systems have important roles in determining airway reactivity, regulating inflammation, and might contribute significantly to NO measured in exhaled breath. METHODS: Bronchial epithelial cells were obtained from healthy, atopic, and asthmatic children by non-bronchoscopic brushing. Exhaled NO (FeNO) was measured directly using a fast response chemiluminescence NO analyser. RNA was extracted from the epithelial cells and real time polymerase chain reaction was used to determine the expression of NOS isoenzymes. NOS2 was examined in macrophages and epithelial cells by immunohistochemistry. RESULTS: NOS1 mRNA was not detectable. NOS3 mRNA was detected in 36 of 43 samples at lower levels than NOS2 mRNA which was detectable in all samples. The median FeNO was 15.5 ppb (95% CI 10 to 18.1). There was a significant correlation between FeNO and NOS2 expression (R = 0.672, p<0.001). All epithelial cells exhibited NOS2 staining, whereas staining in the macrophages was variable and not related to phenotype. CONCLUSIONS: Only NOS2 expression was associated with FeNO in respiratory epithelial cells obtained from children (R = 0.672; p<0.001). This suggests that FeNO variability is largely determined by epithelial NOS2 expression with little contribution from other isoforms.

Regional differences in sympathetic purinergic transmission along the length of the mouse vas deferens.

Contraction of the smooth muscle in the mouse vas deferens is elicited by sympathetic nerves releasing at least two neurotransmitters, adenosine triphosphate (ATP) and noradrenaline (NA). Several studies have indicated the presence of regional variation in the purinergic and noradrenergic contributions to sympathetic nerve-evoked contractions in rodent vasa deferentia. We examined the relative contribution of ATP and NA to neurotransmission and contraction at the prostatic and epididymal ends of the mouse vas deferens. The success rate of recording excitatory junction currents (EJCs, extracellular indication of ATP release) from varicosities at the prostatic end of the vas deferens was eight times greater than for varicosities located at the epididymal end. Both regions of the vas deferens responded similarly to focal application of NA and ATP. Furthermore, the relative density and distribution of P2X(1)-receptor mRNA and anti-P2X(1) immunostaining did not differ between the two regions. Our results suggest that most varicosities located at the epididymal end of the vas deferens are releasing an insufficient amount of ATP to evoke detectable EJCs.

Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma.

BACKGROUND: Protease-activated receptors (PARs), which are G protein-coupled receptors that are activated after proteolytic cleavage of the amino terminus of the receptor, are likely to play a major role in airway inflammation. PARs are activated by endogenous proteases, including thrombin (PAR-1, -3, and -4) and tryptase (PAR-2 and -4), both of which are present in inflamed airways. OBJECTIVE: The purpose of this study was to compare the expression and distribution of PARs in biopsy specimens obtained from asthmatic and normal subjects and to examine the effect of inhaled corticosteroids on PAR expression. METHODS: Biopsy specimens were obtained from 10 normal and 20 asthmatic patients, and sections were stained for PAR-1, -2, -3, and -4 through use of specific antibodies. Staining was scored semiquantitatively for both intensity and distribution. RESULTS: Staining for all PARs was seen on the epithelium and smooth muscle in biopsy specimens from both normal and asthmatic subjects. In the epithelium, PAR-1 and -3 staining appeared to be apically concentrated, whereas PAR-2 and -4 staining was more diffuse. In normal subjects, epithelial staining intensity of PAR-1 and -3 was significantly greater than for PAR-4 (P < .05). Staining for PAR-1, -3, and -4 in biopsy specimens from asthmatic subjects was similar to that in specimens from normal subjects, irrespective of whether the former were using inhaled corticosteroids. However, PAR-2 staining in asthmatic epithelium was significantly increased in comparison with normal epithelium. Expression of PARs in airway smooth muscle did not differ between groups. CONCLUSION: Asthma per se is associated with increased PAR-2 expression in bronchial epithelium. Importantly, staining was not influenced by inhaled corticosteroids. These results suggest that PAR-2 might be involved in airway inflammation.

Activity of an injectable, sustained-release formulation of moxidectin administered prophylactically to mixed-breed dogs to prevent infection with Dirofilaria immitis.

OBJECTIVE: To test the ability of a single injection of a sustained-release formulation of moxidectin (moxidectin SR) to protect dogs against heartworm infection for 180 days after inoculation with infective third-stage larvae (L3) of Dirofilaria immitis. ANIMALS: 32 adult mixed-breed dogs. PROCEDURE: Dogs were allocated to 4 groups on the basis of weight and sex. Dogs were injected SC with saline (0.9% NaCl) solution or moxidectin SR at the rate of 0.06, 0.17, or 0.5 mg/kg of body weight (day 0). Each dog was inoculated SC with 50 D immitis L3 180 days later. On days 330 and 331, dogs were euthanatized. The heart, lungs, and thoracic cavity were examined, and number and sex of heartworms were determined. RESULTS: A mean of 35.9 heartworms was recovered from untreated control dogs. Fourteen worms were recovered from 1 of 8 dogs given moxidectin SR at the lowest dosage, and none of the dogs in the 2 highest moxidectin treatment groups were infected. Small barely palpable granulomas were detected at injection sites of moxidectin-treated dogs. Frequency and size of granulomas were positively correlated with dose of moxidectin administered. CONCLUSIONS AND CLINICAL RELEVANCE: A single dose of moxidectin SR at a dosage as low as 0.17 mg/kg can safely and reliably confer complete protection against infection after challenge-exposure with D. immitis L3, and protection lasts for at least 180 days. This mode of prophylactic treatment against infection with heartworms effectively eliminates failure of prophylaxis that results from erratic administration of medications designed for monthly administration.

Inhibition of angiogenesis-related endothelial activity by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective in the prevention/control of acute and chronic rejection in animal models and currently in phase I clinical trials in human transplant recipients. This agent is also effective in the control of graft-versus-host disease, autoimmune reactions, and the growth of certain tumors. The importance of the endothelium in these disease processes led us to hypothesize that leflunomide might act directly upon the endothelial cell (EC). METHODS AND RESULTS: Assay of human EC colony formation demonstrated dose-dependent, leflunomide-mediated inhibition of EC proliferation. In addition, the organization of EC into capillary-like networks, which occurs during 18 hr of incubation on Matrigel, was progressively disrupted with increasing concentrations of leflunomide. Finally, fibrin-embedded transverse sections of murine aorta, which sprout numerous microvessels during an 11-day incubation, were inhibited from doing so in the presence of this agent. All drug concentrations used in these experiments were nontoxic and pharmacologically relevant, and none of these effects were reversible by exogenous uridine, implying that inhibition of these processes was not due to intracellular pyrimidine depletion. Furthermore, neither cyclosporine nor tacrolimus exerted inhibitory activity in any of the experiments described above. CONCLUSIONS: Data generated by these studies distinguish leflunomide among immunosuppressants as uniquely capable of inhibiting angiogenesis-related endothelial functions and suggest additional mechanisms by which this agent might intervene in the diverse array of disease processes against which it has shown therapeutic potential.