Thursday, 24 April 2014

RSPCA calls for end of MS Animal Research

RSPCA (Royal Society of Prevention of Cruelty to Animals) calls for end to `severe' suffering of lab animals

Charity urges Government to take action on World Day for Laboratory Animals

Today (24 April) is World Day for Laboratory Animals and the RSPCA is calling on the UK Government to commit to ending the use of animals in experiments that cause ‘severe’ suffering.

Scientific procedures using animals in the UK are classified as ‘mild’, ‘moderate’ or ‘severe’, and while any level of suffering is a concern for the RSPCA, ending severe suffering is a top priority.

The amount of pain or distress experienced by each lab animal depends on the kind of research being done, and also on how much effort has been made to reduce or avoid suffering. Each year, animals are involved in experiments categorised as ‘severe’, which cause severe pain, suffering or distress, or ‘severe impairment of their well being or general condition’. The exact number of animals experiencing this will be unknown, until changes in reporting take effect next year - but even a 2% level of severe suffering will involve tens of thousands of animals.

This is a major animal welfare and ethical issue that the RSPCA believes must be addressed as a matter of urgency. We are urging the UK Government to take a policy decision not to license animal experiments in which animals would be expected to experience ‘severe’ suffering.

RSPCA Senior Scientist Dr Penny Hawkins said: “A Government decision not to allow severe suffering would truly focus the minds of those using animals in research, or who fund animal use, on ensuring that absolutely everything possible is done to avoid severe pain, suffering, distress or lasting harm.

“People often say that they work to ‘high welfare standards’ and do not want to cause severe suffering, so they should view a decision not to license these experiments as a welcome challenge to help them make real progress towards more humane science.”

RSPCA calls for end of MS Animal Research. They do not say this directly but this is what they mean as Animal Experiments on Multiple Sclerosis are considered to be "Severe" . Therefore these types of experiments could leave these shores. 

Hurray you may say.....but where does this work move to?....

To places where they hang animals upside down by their tail for months.....?

Exome sequencing

Kemppinen A, Baker A, Liao W, Fiddes B, Jones J, Compston A, Ban M, Sawcer S. Exome sequencing in single cells from the cerebrospinal fluid in multiple sclerosis. Mult Scler. 2014 Apr. [Epub ahead of print]

BACKGROUND Genome-wide association studies (GWAS) have identified over 100 germline variants that influence susceptibility to multiple sclerosis, most of which map within or near to genes with immunological function. However, the role of somatic mutations in multiple sclerosis has not been investigated.

OBJECTIVE:The objective of this paper is to explore the role that somatic mutations might play in the development of multiple sclerosis.

METHODS:We exome-sequenced in total 21 individual CD4+ lymphocytes isolated from cerebrospinal fluid of two patients. In addition we sequenced DNA from the patients' peripheral blood to serve as germline reference.

RESULTS:In comparison with the respective germline sequence, each cell differed at an average of 1784 positions, but as anticipated subsequent analysis confirms that most, if not all, of these potential mutations are likely to represent artefacts generated during the amplification of a single genome and/or by sequencing. Fifty-six of the potential mutations were predicted to have likely functional effects on genes that have previously been implicated by GWAS, including three in the CD6 gene.

CONCLUSION: More robust methods applied to larger numbers of cells will be needed to define the role of somatic mutations.

When DNA is made into a protein the introns (non-coding bits) of DNA are removed to have RNA made up of exons (coding bits) of the DNA, this can be sequenced to see what proteins are likely to be made.

Exome sequencing (also known as targeted exome capture) is an efficient strategy to selectively sequence the coding regions of the genome as a cheaper but still effective alternative to whole genome sequencing. Exons are short, functionally important sequences of DNA which, together, represent only slightly more than the portion of the genome that is actually translated into protein. Exons are flanked by untranslated regions (UTR) that are usually not included in exome studies. In the human genome there are about 180,000 exons. These constitute about 1% of the human genome.

They were looking for mutations (switches of the original DNA sequence) when CD4 T cells made protein but the was loads of mistakes in the technology, so the technology needs improving. The may have found some in CD6. What does it mean I don't know?, but this introduces you to the idea of technology. 

So once every body has been genome-sequenced we can do it again and again for every, cell type at different time points.

A phenome is the set of all phenotypes expressed by a cell, tissue, organ, organism, or species. Just as the genome and proteome signify all of an organism's genes and proteins, the phenome represents the sum total of its phenotypic traits. Examples of human phenotypic traits are skin color, eye color, body height, or specific personality characteristics. Although any phenotype of any organism has a basis in its genotype, phenotypic expression may be influenced by environmental influences, mutation, and genetic variation such as single nucleotide polymorphisms (SNPs), or a combination of these factors.

Phenomics is the study of the phenome and how it is determined, particularly when studied in relation to the set of all genes (genomics) or all proteins (proteomics).

Carroll RJ, Bastarache L, Denny JC. R PheWAS: Data Analysis and Plotting Tools for Phenome Wide Association Studies in the R Environment. Bioinformatics. 2014 Apr . [Epub ahead of print]

Anti-JC virus antibodies in CSF and PML

Natalizumab is not 100% effective in stopping immune responses within the CNS #MSBlog #MSResearch

"The following study uses a very old technique of looking for the production of specific antibodies within the central nervous system (CNS), or intrathecal compartment (within the membranous coverings of the brain), to help diagnose PML. The investigators used a so called JCV antibody index to detect antibody production within the CNS against the JC virus. The way this technique works is that they have to normalise the levels of anti-JCV antibodies against a ratio of other proteins to account for a leaky blood brain barrier, which is the norm in MSers. This correction allows you to create a virus specific antibody index; an index greater than 1.5 indicates that there is production of anti-JCV antibodies within the CNS. It implies that there are B cells and plasma cells in  the brain and spinal cord that are making anti-JCV antibodies. We know that this occurs commonly in other viral infections of the brain, for example with mumps, measles, rubella and herpes infections. The investigators found that in 70% of MSers who had PML diagnosed using MRI and the detection of virus specific DNA had an index greater than 1.5. This means that 30% did not. In general a diagnostic test has to be at least 80% sensitive (detecting PML) and specific (excluding PML) before it can be used clinically. I am therefore not sure about their conclusion that the index can be used to help diagnose PML. More work needs to be done on this assay. A factor that needs to be considered is the mode of action of natalizumab; it blocks lymphocyte trafficking into the CNS therefore it potentially could prevent and adequate CNS B-cell response which may explain why the only 70% had an index > 1.5 and not a figure closer to 100%. The good news is that their data suggests that there is some immune response to the virus in MSers with PML on natalizumab. This indicates that natalizumab is not 100% effective at blunting the immune responses within the CNS and may explain why some MSers with PML have low-grade immune reconstitution inflammatory syndrome (IRIS) despite still being on natalizumab and that most biopsies done when MSers are on natalizumab show inflammatory cell infiltrates. This data suggests we may need more effective cell trafficking blockers that natalizumab to treat MS. This is the first time I have thought about actually improving on the efficacy that natalizumab provides. Interesting?"

Epub: Warnke et al. The CSF JCV antibody index for diagnosis of natalizumab-associated PML. Ann Neurol. 2014 Apr . doi: 10.1002/ana.24153.

Background: Progressive multifocal leukoencephalopathy (PML), caused by JC virus (JCV), can occur in MSers receiving natalizumab for MS. JCV detection by quantitative polymerase chain reaction (qPCR) in cerebrospinal fluid (CSF), or brain biopsy is required for probable or definite diagnosis of PML. However, in some MSers only low levels of JCV-DNA (<100 copies/ml) are present in CSF, making the diagnosis challenging. 

Objective: To assess the complementary value of a CSF JCV antibody index (AIJCV ) in the diagnosis of natalizumab-associated PML. 

Methods: In 37 cases of natalizumab-associated PML and 89 MSers treated with natalizumab without PML AIJCV was assessed. Sera and CSF were tested in a capture ELISA, using JCV-VP1 fused to glutathione S-transferase (GST) as antigen. Albumin levels and total IgG concentration were determined by immunonephelometry, and the AIJCV was calculated as published. 

Results: Twenty-six of 37 (70%) patients with natalizumab-associated PML exhibited an AIJCV >1.5, while this was seen in none of the controls (p<0.0001). At time of the first positive qPCR for JCV-DNA, 11/20 (55%) of MSers with natalizumab-associated PML had an AIJCV >1.5. JCV-DNA levels of <100 copies/ml were seen in 14/20 (70%) of these MSers of which 8 (57%) demonstrated an AIJCV >1.5. 

Interpretation: Determination of the AIJCV could be an added tool in the diagnostic workup for PML and should be included in the case definition of natalizumab-associated PML.

CoI: multiple