Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti-neutrophil cytoplasmic antibodies.

Anti-neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA-associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3-ANCA-positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P = 0·019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N-terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.

Epitope shift of proteinase-3 anti-neutrophil cytoplasmic antibodies in patients with small vessel vasculitis.

Anti-neutrophil cytoplasmic antibodies against proteinase 3 (PR3-ANCA) are used as diagnostic tools for patients with small vessel vasculitis (AASV). We have produced chimeric mouse/human PR3 molecules and investigate changes in reactivity over time and the possible relationship between epitope specificity and clinical course. Thirty-eight PR3-ANCA-positive patients diagnosed between 1990 and 2003 were followed until December 2005. Plasma was collected at each out-patient visit and older samples were retrieved retrospectively. Patients reacted with multiple epitopes at the time of diagnosis. At subsequent relapses 12 patients shifted reactivity, in 11 cases from epitopes located in the C-terminal towards epitopes in the N-terminal. Patients with reactivity against N-terminal parts of PR3 at diagnosis had a significantly lower relapse rate, 30% compared to 78% in the group with predominantly C-terminal reactivity (P = 0.04). The reactivity pattern did not correlate to outcome measured as death, end-stage renal disease or vasculitis activity index score (VDI) at 5 years. Further research is necessary to conclude if this is a general phenomenon.

Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression.

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Ultrahigh sensitivity made simple: nanoplasmonic label-free biosensing with an extremely low limit-of-detection for bacterial and cancer diagnostics.

We present a simple and robust scheme for biosensing with an ultralow limit-of-detection down to several pg cm(-2) (or several tens of attomoles cm(-2)) based on optical label-free biodetection with localized surface plasmon resonances. The scheme utilizes cost-effective optical components and comprises a white light source, a properly functionalized sensor surface enclosed in a simple fluidics chip, and a spectral analyzer. The sensor surface is produced by a bottom-up nanofabrication technique with hole mask colloidal lithography. Despite its simplicity, the method is able to reliably detect protein-protein binding events at low picomolar and femtomolar concentrations, which is exemplified by the label-free detection of the extracellular adherence protein (EAP) found on the outer surface of the bacterium Staphylococcus aureus and of prostate-specific antigen (PSA), which is believed to be a prostate cancer marker. These experiments pave the way towards an ultra-sensitive yet compact biodetection platform for point-of-care diagnostics applications.

A carbohydrate binding module as a diversity-carrying scaffold.

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

The mortality of motor neuron disease in Sweden.

The age-standardized mortality from motor neuron disease in Sweden doubled from 1961 to 1985. The average annual rate during the period was 1.9 per 100,000 population. The male to female ratio was 1.2:1. The age-specific mortality rates had a peak at 70 to 79 years of age. When each birth cohort was followed up separately over time, the peak was less clear and in some cohorts the mortality rates increased continuously with advancing age. A significant increase of motor neuron disease among men was found in one Swedish county.

Respiratory function, electrocardiography and quality of life in individuals with muscular dystrophy.

All individuals in a Swedish county afflicted with any type of hereditary muscular dystrophy (MD) were identified and 57 (85 percent) of eligible individuals in the age range 16 to 64 were included in the study. Respiratory disturbances were estimated by means of spirometry and analysis of arterial blood gases, and 58 percent yielded abnormal results on at least one of these examinations. Elevated PCO2 was found more commonly than reduced forced vital capacity (FVC) and there was a moderate association between these parameters. Respiratory symptoms, most commonly breathlessness, were encountered in 79 percent. Pathologic ECG recordings were found in 21 individuals (37 percent). Conduction disturbances and affection of the myocard were most frequent in myotonic dystrophy. Quality of life was assessed by means of the Sickness Impact Profile instrument and the Kaasa test. The results showed that quality of life was significantly related to FVC and to the symptom of abnormal fatigue. Respiratory and cardiac parameters showed a greater number of significant correlations with measures of functional ability than with subjective well-being.

In vitro lymphoproliferative assays with HgCl2 cannot identify patients with systemic symptoms attributed to dental amalgam.

Dental amalgam is suspected, by some exposed individuals, to cause various systemic psychological, sensory, and neurological symptoms. Since not all amalgam-bearers experience such reactions, an individual characteristic--for example, a susceptible immune system--might explain these conditions. In vitro lymphocyte proliferation is a valuable tool in the diagnosis of allergy. With HgCl2 as the antigen, however, the test is hampered, because Hg2+ can cause unspecific lymphocyte proliferation, optimal at 1.4 to 9.5 micrograms HgCl2/mL. Recently, the use of suboptimal HgCl2 concentrations (< or = 0.5 microgram/mL) has been suggested to circumvent these problems. The main aim of this study was to investigate whether patients with systemic symptoms alleged to result from the presence of dental amalgam differ from healthy controls, with reference to in vitro lymphoproliferative responses to HgCl2 < or = 0.5 microgram/mL. Three different test protocols--lymphocyte transformation test (LTT) in micro- and macro-cultures, and the memory lymphocyte immunostimulation assay (MELISA)--were used. Other immune parameters--such as a standard patch test for dental materials, the number of T- and B-lymphocytes, monocytes, granulocytes, and NK cells in peripheral blood, allergic symptoms, and predisposition--were also investigated. Twenty-three amalgam patients, 30 healthy blood donors with amalgam, ten healthy subjects without amalgam, and nine patients with oral lichen planus (OLP) adjacent to dental amalgam and a positive patch test to Hg0 were tested. None of the investigated immune parameters revealed any significant differences between amalgam patients and controls. The sensitivity of in vitro lymphocyte proliferation ranged from 33 to 67%, with the OLP patients as a positive control group, and the specificity from 0 to 70% for healthy controls with a negative patch test to Hg0. Thus, despite the use of HgCl2 < or = 0.5 microgram/mL, a high frequency of positive results was obtained among healthy subjects with or without dental amalgam. Consequently, in vitro lymphocyte proliferation with HgCl2 cannot be used as an objective marker for mercury allergy in dental amalgam-bearers.

Motor neuron disease and exposure to chemicals--aetiological suggestions from a case-control study.

A recent case-control study indicates that some males are genetically predisposed to develop motor neuron disease if they are exposed to chemicals, particularly solvents. Markers for such a predisposition are hereditary for a neurodegenerative disease and/or a thyroid disease. The reason for this susceptibility might be pharmacogenetic polymorphism, which causes deficient detoxification entailing oxidative stress and cell death.

Serotonin production in lymphocytes and mercury intolerance.

Patients with suspected illness due to mercury in dental amalgam were classified as tolerant or intolerant depending on their psychosomatic responses following in vivo epicutaneous provocation with low doses (patch test doses) of metallic mercury and phenylmercuric acetate. Ten intolerant patients and nine tolerant patients plus seven healthy amalgam-free and metal non-allergic controls were recruited to the study. Peripheral blood lymphocytes were exposed in vitro to three concentration of mercuric chloride (0.92, 1.83 and 3.68 microM) with and without 10 microg phytohaemagglutinine (PHA)/ml and the release of serotonin into the supernatant was measured. Lymphocytes exposed only to HgCl(2) showed no significant dose-dependent increase of serotonin, but the response of the tolerant patients was significantly higher compared with the controls. No other differences were found. Co-culture with mercuric chloride and PHA showed a statistically significant dose-dependant release of serotonin, but no differences between the three clinical groups could be detected. Thus, our results could not validate the concept of mercury tolerance and intolerance.

Demonstration of circulating antibodies to Pneumonyssoides caninum in experimentally and naturally infected dogs.

Antibodies to the canine nasal mite, Pneumonyssoides caninum, were demonstrated by ELISA in sera from four experimentally and 77 naturally infected dogs employing a crude P. caninum antigen. In sera from the four experimentally infected dogs, antibodies to P. caninum were first detected on day 11 post-inoculation (p.i.). Optical density (OD) values remained high throughout the observation period of 14 weeks. Two major protein bands with apparent molecular weights of 83 and 74kDa were visible by western blot (WB) from day 11 p.i. On day 14 p.i., another three major protein bands at 44, 37, and 34kDa were visible. The protein pattern then remained essentially constant until the end of the experiment except for some weak bands between 250 and 30kDa which were sometimes visible. In sera obtained from a non-inoculated control dog, no P. caninum antibodies were detected, neither by the ELISA nor by the WB. With sera from 77 naturally infected dogs with a verified diagnosis of P. caninum infection, the OD values varied between 0.24 and 0.95. When sera from four of these dogs were analysed by WB, the protein pattern observed was equivalent with that seen with sera from the experimentally infected dogs on day 25 p.i. Sera from four experimental Beagles, in which no P. caninum mites were found at necropsy, were also analysed by the P. caninum ELISA, the OD values demonstrated never exceeded 0.15. In addition, 48 sera obtained from dogs infected with or allergic to other arthropods were used for studies of possible cross-reactivity. With 10 sera, elevated OD values were found by the ELISA. Eight of them showed a similar banding pattern by the WB as did the experimentally infected dogs on day 25 p.i. This was, therefore, interpreted as an effect of a concurrent P. caninum infection.

Experimental infection of dogs with the nasal mite Pneumonyssoides caninum.

A successful experimental transmission of the canine nasal mite, Pneumonyssoides caninum, is described. Some 11 weeks after repeated systemic ivermectin treatment, four Beagles were inoculated via the right nostril with 20 P. caninum mites of different sexes and life stages, obtained at the necropsy of an infected dog. The inoculated dogs and a matching uninoculated control were observed for clinical signs for 14 weeks and then euthanised. Vague upper respiratory signs and a transient minor increase in the number of eosinophils in peripheral blood were recorded in the inoculated dogs. At necropsy 4-12 P. caninum mites were found in the nasal cavities and sinuses of the inoculated dogs, but none in the control. In three out of the four infected dogs mites were found in both the right and left nasal cavities and sinuses of the skull. Since in no case more mites than the number used for inoculation were detected it is not clear if the mites managed to reproduce in the dogs. Inflammatory lesions were seen most consistently in the olfactory mucosa, respiratory mucosa and tonsils, and growth of opportunistic bacteria was observed in the tonsils of the infected dogs. The inflammatory lesions seen in the olfactory mucosa may explain why dogs infected with P. caninum sometimes appear to suffer from impaired scenting ability.

Disability, coping and quality of life in individuals with muscular dystrophy: a prospective study over five years.

PURPOSE: The present study investigates progressive muscular dystrophy over a five year period. The purpose is twofold: to describe changes over time and to investigate relations between disability, coping and quality of life. METHOD: The study group comprised 45 adults (16 men and 29 women), with an average age of 44 years. All were assessed in 1991 and 1996, with the following instruments: the ADL staircase, the Self-report ADL, the Mental Adjustment to Cancer scale, the Sickness Impact Profile and the Psychosocial well-being questionnaire (Kaasa). RESULTS: Increasing disability was accompanied by an increase in dependence on others and a significant deterioration of health-related quality of life and with regard to 'Satisfaction'. The predominant type of coping was 'Fighting spirit', whilst 'Fatalism' showed the greatest decline over time. 'Ambulation' and the ADL staircase correlated with 'Physical index' on the SIP. Correlations between disability, coping and quality of life were moderate. The results can serve as a basis for planning and evaluation of recurring rehabilitation for persons with MD.

The diagnosis of carpal tunnel syndrome. Sensitivity and specificity of some clinical and electrophysiological tests.

The study group consisted of 100 persons referred with suspected carpal tunnel syndrome. Clinical and neurophysiological examinations were performed blinded from each other. The gold standard for the carpal tunnel syndrome (CTS) diagnosis was based on the results of these examinations but relief of CTS symptoms after surgery was also required. The sensitivity and specificity for the combined results of the clinical examinations were 94% and 80% respectively, and for the neurophysiological examinations, 85% and 87%. Of the neurophysiological methods used, the quotient of sensory nerve conduction velocity between palm to wrist and wrist to elbow was best and the cut-off for this test was studied by means of an ROC-curve. According to our results clinical examination by an experienced doctor seems to be sufficient if there are typical symptoms of carpal tunnel syndrome, but if there is a history of pain, atypical symptoms or earlier fractures in the arm, wrist or hand, it is important to add a neurophysiological examination.

Diseases associated with pronounced eosinophilia: a study of 105 dogs in Sweden.

Records of 105 dogs with pronounced eosinophilia (>2.2 x 10(9) eosinophils/litre) were evaluated in a retrospective study to determine diseases associated with the abnormality in dogs in Sweden. Inflammatory disease in organs with large epithelial surfaces, such as the gut, lungs or skin, was found in 36 per cent of the dogs. A further one-quarter of the 105 cases were placed in the 'miscellaneous' category, which comprised various diseases found at low frequency. The most well defined diagnosis was pulmonary infiltrates with eosinophils in 12 per cent of the dogs. A further 11 per cent had parasitic disease caused by either sarcoptic mange or nasal mite. No atopic dog was found and rottweilers were over-represented in most disease groups. Pronounced eosinophilia, in many cases transient, seems to be associated with a variety of disorders in dogs. In the present study, rottweilers appeared to be more prone to a high eosinophil response than other breeds.

Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology.

Fish represent the planet's most diverse group of vertebrates and they can be exposed to a wide range of pharmaceuticals. For practical reasons, extrapolation of pharmaceutical effects from 'model' species to other fish species is adopted in risk assessment. Here, we critically assess this approach. First, we show that between 65% and 86% of human drug targets are evolutionarily conserved in 12 diverse fish species. Focusing on nuclear steroid hormone receptors, we further show that the sequence of the ligand binding domain that plays a key role in drug potency is highly conserved, but there is variation between species. This variation for the oestrogen receptor, however, does not obviously account for observed differences in receptor activation. Taking the synthetic oestrogen ethinyloestradiol as a test case, and using life-table-response experiments, we demonstrate significant reductions in population growth in fathead minnow and medaka, but not zebrafish, for environmentally relevant exposures. This finding contrasts with zebrafish being ranked as more ecologically susceptible, according to two independent life-history analyses. We conclude that while most drug targets are conserved in fish, evolutionary divergence in drug-target activation, physiology, behaviour and ecological life history make it difficult to predict population-level effects. This justifies the conventional use of at least a 10× assessment factor in pharmaceutical risk assessment, to account for differences in species susceptibility.

Prioritising pharmaceuticals for environmental risk assessment: Towards adequate and feasible first-tier selection.

The presence of pharmaceuticals in the aquatic environment, and the concerns for negative effects on aquatic organisms, has gained increasing attention over the last years. As ecotoxicity data are lacking for most active pharmaceutical ingredients (APIs), it is important to identify strategies to prioritise APIs for ecotoxicity testing and environmental monitoring. We have used nine previously proposed prioritisation schemes, both risk- and hazard-based, to rank 582 APIs. The similarities and differences in overall ranking results and input data were compared. Moreover, we analysed how well the methods ranked seven relatively well-studied APIs. It is concluded that the hazard-based methods were more successful in correctly ranking the well-studied APIs, but the fish plasma model, which includes human pharmacological data, also showed a high success rate. The results of the analyses show that the input data availability vary significantly; some data, such as logP, are available for most API while information about environmental concentrations and bioconcentration are still scarce. The results also suggest that the exposure estimates in risk-based methods need to be improved and that the inclusion of effect measures at first-tier prioritisation might underestimate risks. It is proposed that in order to develop an adequate prioritisation scheme, improved data on exposure such as degradation and sewage treatment removal and bioconcentration ability should be further considered. The use of ATC codes may also be useful for the development of a prioritisation scheme that includes the mode of action of pharmaceuticals and, to some extent, mixture effects.