Application of congener based multi-matrix profiling techniques to identify potential PCDD/F sources in environmental samples from the Burrishoole Catchment in the West of Ireland.

Homologue and congener profiles of PCDD/Fs in eels, passive sampler and sediment extracts from the Burrishoole, a rural upland catchment on the western Irish seaboard were compared with potential PCDD sources. ΣPCDD/F levels in eels ranged from 2.9 to 25.9 pg g(-1) wet weight, which are elevated compared to other Irish locations. The OCDD congener dominated the pattern of ΣPCDD/Fs in all matrices from Burrishoole. Passive samplers were successfully deployed to identify for the first time the presence in the water column of PCDD/Fs and dimethoxylated octachlorodiphenyl ether (diMeOoctaCDE), impurities found in pentachlorophenol (PCP) production. Principal component analysis (PCA) identified similarities between PCDD/F profiles in technical PCP mixtures and environmental samples from the Burrishoole region. Results strongly suggest residual PCDD contamination associated with historic local use of a dioxin contaminated product in the catchment area, with pentachlorophenol a strong candidate.

Determination of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry at cross contamination levels.

A confirmatory multi-residue method has been developed to allow for the detection, confirmation and quantification of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method can be used to determine halofuginone, robenidine, nicarbazin, diclazuril, decoquinate, semduramicin, lasalocid, monensin, salinomycin, narasin, maduramicin at levels relating to unavoidable carry over as stated in Regulation 2009/8/EC. Feed samples are extracted with water and acetonitrile with the addition of anhydrous magnesium sulphate and sodium chloride. The extract then undergoes a freezing out step before being diluted and injected onto the LC-MS/MS system. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation and can confirm all eleven analytes in a run time of 19 min. The sensitivity of the method allows quantification and confirmation for all coccidiostats at a 0.5% carry over level. The method was validated over three days in accordance with of European legislation; Commission Decision 2002/657/EC. Validation criteria of accuracy, precision, decision limit (CCα), and detection capability (CCß) along with measurement uncertainty are calculated for all analytes. The method was then successfully used to analyse a number of feed samples that contained various coccidiostat substances.

Development and validation of a rapid multi-class method for the confirmation of fourteen prohibited medicinal additives in pig and poultry compound feed by liquid chromatography-tandem mass spectrometry.

A confirmatory method has been developed to allow for the analysis of fourteen prohibited medicinal additives in pig and poultry compound feed. These compounds are prohibited for use as feed additives although some are still authorised for use in medicated feed. Feed samples are extracted by acetonitrile with addition of sodium sulfate. The extracts undergo a hexane wash to aid with sample purification. The extracts are then evaporated to dryness and reconstituted in initial mobile phase. The samples undergo an ultracentrifugation step prior to injection onto the LC-MS/MS system and are analysed in a run time of 26 min. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation. The method was validated over three days and is capable of quantitatively analysing for metronidazole, dimetridazole, ronidazole, ipronidazole, chloramphenicol, sulfamethazine, dinitolimide, ethopabate, carbadox and clopidol. The method is also capable of qualitatively analysing for sulfadiazine, tylosin, virginiamycin and avilamycin. A level of 100 microg kg(-1) was used for validation purposes and the method is capable of analysing to this level for all the compounds. Validation criteria of trueness, precision, repeatability and reproducibility along with measurement uncertainty are calculated for all analytes.

Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography-tandem mass spectrometry (LC-MS).

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 microg l(-1)/microg kg(-1) for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 microg l(-1)/microg kg(-1) is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCalpha) and detection capabilities (CCbeta) ranged from 0.41 to 1.55 microg l(-1) and from 0.70 to 2.64 microg l(-1), respectively, in milk; and from 0.38 to 1.16 microg kg(-1) and from 0.66 to 1.98 microg kg(-1), respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 microg l(-1) in milk and 0.08 and 0.13 microg kg(-1) in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.

Myasthenic crisis.

Myasthenic crisis is a life-threatening medical emergency requiring early diagnosis and respiratory assistance. It can affect between one-fifth and one-third of all patients with generalized autoimmune myasthenia gravis. Myasthenic crisis is to be distinguished from other causes of acute neuromuscular paralysis which in most cases, can be achieved clinically. High dose corticosteroids in combination with plasma exchange or immunoglobulin are the cornerstone of treatment for this fully reversible cause of neuromuscular paralysis.

Treatment of multiple sclerosis: beyond the NICE guidelines.

Multiple sclerosis (MS) is a common, disabling neurological condition whose pathogenesis is not clearly understood. Although current treatment recommendations assume an immunopathogenic disease mechanism, MS may not be an autoimmune disorder. Long-term immunological therapy for MS is in our view an untested approach, guided by uncritical acceptance of data from drug trials. We do not believe that there is convincing evidence that any of these immune-based treatments prevents long-term disease progression, or has much effect on common disabilities such as fatigue, pain, depression and cognitive impairment. The recent recommendations of the National Institute of Clinical Excellence did not address important issues regarding disease modification, management of paroxysmal symptoms and the likely therapeutic candidates for future treatment trials. We discuss treatment options for MS beyond the NICE guidelines.

In vivo magnetic resonance spectroscopy in chronic fatigue syndrome.

The pathogenic mechanisms of chronic fatigue syndrome (CFS) are not clearly known. Fatigue, poor short-term memory and muscle pain are the most disabling symptoms in CFS. Research data on magnetic resonance spectroscopy (MRS) of muscles and brain in CFS patients suggest a cellular metabolic abnormality in some cases. 31P MRS of skeletal muscles in a subset of patients indicate early intracellular acidosis in the exercising muscles. 1H MRS of the regional brain areas in CFS have shown increased peaks of choline derived from the cell membrane phospholipids. Cell membrane oxidative stress may offer a common explanation for the observed MRS changes in the muscles and brain of CFS patients and this may have important therapeutic implications. As a research tool, MRS may be used as an objective outcome measure in the intervention studies. In addition, regional brain 1H MRS has the potential for wider use to substantiate a clinical diagnosis of CFS from other disorders of unexplained chronic fatigue.

Molecular mimicry in type 1 diabetes: immune cross-reactivity between islet autoantigen and human cytomegalovirus but not Coxsackie virus.

Type 1 diabetes is caused by a T cell-mediated autoimmune destruction of the pancreatic beta cells. Molecular mimicry between viral pathogens and beta cell protein has been a popular theory to explain loss of tolerance in type 1 diabetes. However, functional data in support of this hypothesis have been lacking, presumably because the homologies were defined on the basis of linear similarities in peptide sequences, which ignores the criteria of HLA versus T cell receptor contact residues in peptide epitopes required for T cell recognition. We applied a HLA-binding dedicated peptide microarray analysis using autoreactive T cell clones specific for the autoantigen GAD65 to determine the algorithm of T cell recognition by this given T cell clone. The subsequent database search identified a 100% fit with cytomegalovirus peptide, which was subsequently shown to be recognized by these clonal T cells. However, T cell clones reactive with linear homologies previously described as putative candidates for T cell cross-reactivity between GAD65 and Coxsackie virus peptide were unable to recognize the homologous counterparts.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase.

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiff-man syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4(+) T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

Acute cervical hyperextension-hyperflexion injury may precipitate and/or exacerbate symptomatic multiple sclerosis.

We report here 39 cases in which definite multiple sclerosis (MS) was precipitated or exacerbated by specific hyperextension-hyperflexion cervical cord trauma. The worsening or onset of the symptomatic disease bore a striking temporal relationship to the focal injury. Our data suggests that central nervous system (CNS)-specific acute physical trauma such as cervical cord hyperextension-hyperflexion injury may aggravate latent clinical symptoms in MS. The deterioration of MS bore no direct relationship with the severity of neck injury. Possible pathogenic mechanisms of focal CNS-specific trauma aggravating the course of asymptomatic or benign MS are discussed. This may have implications in improving our understanding of the factors that may modify the clinical course of MS.

DXA body composition studies are not affected by extracellular water measurements using stable sodium bromide dilution.

Body composition studies using dual energy x-ray absorptiometry (DXA) are being increasingly reported in the literature. When DXA body composition measurements are combined with body water studies, stable bromide is often administered to measure extracellular water. Bromine attenuates x-rays significantly more than soft tissue and so could affect DXA body composition analysis. DXA scans were performed on 26 adults (12 F, 14 M) before and after the intravenous injection of 3 g sodium bromide (NaBr). No significant differences were noted pre- and post-NaBr infusion for whole-body fat mass, fat-free soft tissue mass and bone mineral content. These findings were supported by a simple mathematical analysis of the likely effect of the sodium bromide infusion. This showed that when 3 g NaBr was introduced into the body, the effect on fat mass estimates was expected to be marginally less than the precision of the DXA technique.

Fatigue and basal ganglia.

Fatigue is a common symptom in neurology and occurs in the diseases of the central and peripheral nervous system. In order to understand the mechanism of fatigue, it is important to distinguish symptoms of peripheral neuromuscular fatigue from the symptoms of physical and mental fatigue characteristic of disorders like Parkinson's disease or multiple sclerosis. We have introduced and defined the concept of central fatigue for the latter disorders. We have further proposed, with supportive neuropathological data, that central fatigue may occur due to a failure in the integration of the limbic input and the motor functions within the basal ganglia affecting the striatal-thalamic-frontal cortical system.

The symptoms of chronic fatigue syndrome are related to abnormal ion channel function.

The pathogenesis of chronic fatigue syndrome (CFS) is unknown but one of the most characteristic features of the illness is fluctuation in symptoms which can be induced by physical and/or mental stress. Other conditions in which fluctuating fatigue occurs are caused by abnormal ion channels in the cell membrane. These include genetically determined channelopathies, e.g. hypokalemic periodic paralysis, episodic ataxia type 2 and acquired conditions such as neuromyotonia, myasthenic syndromes, multiple sclerosis and inflammatory demyelinating polyneuropathies. Our hypothesis is that abnormal ion channel function underlies the symptoms of CFS and this is supported also by the finding of abnormal cardiac-thallium201 SPECT scans in CFS, similar to that found in syndrome X, another disorder of ion channels. CFS and syndrome X can have identical clinical symptoms. CFS may begin after exposure to specific toxins which are known to produce abnormal sodium ion channels. Finally, in CFS, increased resting energy expenditure (REE) occurs, a state influenced by transmembrane ion transport. The hypothesis that ion channels are abnormal in CFS may help to explain the fluctuating fatigue and other symptoms.

The effect of anti-alpha4 integrin antibody on brain lesion activity in MS. The UK Antegren Study Group.

OBJECTIVE: To determine the effect of humanized monoclonal antibody against alpha4 integrin (reactive with alpha4beta1 integrin or very-late antigen-4) on MRI lesion activity in MS. METHODS: A randomized, double-blind, placebo-controlled trial in 72 patients with active relapsing-remitting and secondary progressive MS was performed. Each patient received two IV infusions of anti-alpha4 integrin antibody (natalizumab; Antegren) or placebo 4 weeks apart and was followed up for 24 weeks with serial MRI and clinical assessment. RESULTS: The treated group exhibited significantly fewer new active lesions (mean 1.8 versus 3.6 per patient) and new enhancing lesions (mean 1.6 versus 3.3 per patient) than the placebo group over the first 12 weeks. There was no significant difference in the number of new active or new enhancing lesions in the second 12 weeks of the study. The number of baseline-enhancing lesions (i.e., lesions that enhanced on the baseline scan) that continued to enhance 4 weeks following the first treatment was not significantly different between the two groups. The number of patients with acute MS exacerbations was not significantly different in the two groups during the first 12 weeks (9 in the treated group versus 10 in placebo) but was higher in the treatment group in the second 12 weeks (14 versus 3; p = 0.005). The study was not, however, designed to look definitively at the effect of treatment on relapse rate. Treatment was well tolerated. CONCLUSIONS: Short-term treatment with monoclonal antibody against alpha4 integrin results in a significant reduction in the number of new active lesions on MRI. Further studies will be required to determine the longer term effect of this treatment on MRI and clinical outcomes.

GAD65-Reactive T cells in a non-diabetic stiff-man syndrome patient.

GAD65 (glutamic acid decarboxylase) is an important autoantigen in both type 1 (insulin-dependent) diabetes mellitus (IDDM) and the neurological autoimmune disease stiff-man syndrome (SMS), and is expressed in pancreatic islets as well as the nervous system. Still, only 30% of SMS patients also have type 1 diabetes. To study regulation of T cell responsiveness to GAD65, we investigated a non-diabetic SMS patient with HLA-DR3/7 (predisposing to type 1 diabetes) and high levels of type 1 diabetes-associated autoantibodies against GAD65 and islet cells, and compared the results with those of her diabetic son and two other SMS patients. T cell responses to GAD65 were repeatedly absent in primary stimulation, whereas IA-2, islet antigen and tetanus toxoid induced significant T cell proliferation. However, after in vitro restimulation, GAD65 reactive T cell lines and clones were obtained that were HLA-DR3 restricted, and cross-reactive with a homogenate of purified human pancreatic islets. These T cells produced the immunoregulatory cytokine IL-10 in combination with IFN-gamma and IL-4 (Th0). The dominant T cell epitope was mapped to the central region of GAD65. Although no primary response to whole GAD65 was detectable, the naturally processed GAD65 peptide epitope was recognized vigorously in the primary stimulation assay. The lack of detectable primary T cell responses to GAD65, together with the GAD65-specific cytokine production of restimulated T cells, suggest that GAD65-specific cellular autoimmunity in this patient is suppressed and may be related to the absence of diabetes despite humoral autoreactivity and genetic predisposition.