Myxovirus Resistance Protein A (MxA) & Antibodies to Nuclear Matrix Protein-2 (NXP-2) in Dermatomyositis Sine Dermatitis

 There has long been debate on what is the most sensitive and specific marker that distinguishes Dermatomyositis (DM) from Polymyositis (PM) or Inclusion Body Myositis (IBM), and further whether anti-synthestase syndromes (ASS) should be included under DM. 

Where subjects present with a skin rash and muscle weakness with one of 5 DM specific autoantibodies, i.e. Mi-2, NXP-2, MDA-5, TIF-1gamma or SAE, the diagnosis of DM is straightforward. Given the difficulty in obtaining muscle biopsies within the NHS, particularly in DGHs, most Rheumatologists would settle for a diagnosis of DM in this scenario, particularly if the EMG too is characteristic of a myopathy.

But what if DM presents without a skin rash, as it can in approximately 8% of subjects. The distinction between DM one one hand and PM, IBM and ASS on the other is more than academic, firstly because of a higher association of cancer with the former, and secondly because of increasing reports of efficacy of JAK inhibitors for DM but not the other conditions.

In this situation, two tests can be useful. The first requires a muscle biopsy, and the second is included in the DM specific autoantibody panel.

First, the staining of the sarcoplasm of muscle for Myxovirus Resistance Protein A (MxA) is highly sensitive (76%) and 100% specific for DM. This is a Type I interferon induced protein and therefore an interferon signature. It is not seen in PM, IBM or ASS. Unlike the latter three, DM is an interferonopathy (which is why it responds to JAK inhibitors).

While other interferon signatures such as RIG-1 and ISG-15 are also quite specific for DM, as indeed are muscle biopsy findings of perifascicular atrophy (PFA) and deposition of membrane attack complex on capillaries, none of these are as sensitive as MxA. (While ASS also displays PFA on biopsy, the characteristic necrotic and regenerating fibres in perifascicular fibres sets it apart from the mainly degenerative fibres seen in DM).

The other useful marker of Dermatomyositis sine (without) dermatitis is the muscle specific antibody NXP-2. This is seen in fully 86% of subjects who have DM without skin involvement at presentation, but only in 28% of subjects who have DM with rash. Therefore, a subject presenting with myositis, but no rash and a positive NXP-2 should be treated as DM rather than PM. In a minority of such subjects, a typical rash may appear after many months or even years.

DM sine dermatitis should not be confused with amyopathic DM, which is characterised by rash and lung involvement without muscle involvement. Most such subjects are positive for MDA-5 and have a severe lung phenotype.

References

1. https://jamanetwork.com/journals/jamaneurology/fullarticle/2764337

2. https://pubmed.ncbi.nlm.nih.gov/30267437/

The New VUI-202012/01 COVID-19 Variant Found in the United Kingdom

 The UK has just tightened its COVID tiers based on a fast spreading variant of the virus picked up by the COVID-19 Genomic Consortium. So far 1108 cases with this variant has been described. Apparently it is 70% more infectious than the prevalent D614G strain. Several European countries have imposed a summary bans on flights originating in the UK as a result.

It is fair to say that as a RNA virus, COVID-19 mutates continuously. A WHO analysis found that the rate of mutations for COVID-19 is 1.12 x 10^-3 mutations per site year, which is quite similar to the range of 0.80 x !0^-3 to 2.38 x 10^-3 per site year for the original SARS virus in 2003.

To put this into some context, the mutation rate in human beings is 1.2 x 10^-8 per generation. This translates to around 72 new mutations in a newborn. Mutagenesis is therefore an inevitable vicissitude of the genetic code.

You may be aware that the current circulating clade- D614G- originated in China in January 2020 and replaced the existing clade within 3 months. It is likely that VUI-202012/01 will become the dominant strain if allowed to spread.

Concern about emergence of new strains is reflected in the Danish government's recent decision to cull 17 million minks because they harboured a variant of the virus that was apparently not well neutralised by existing human antibodies.

The new variant VUI-202012/01 has 17 new mutations- the most important of which is N501Y involving the spike protein (which means that tyrosine has replaced asparagine in the 501st amino acid of the spike protein).

Looking at the DNA code, asparagine has 2 codons- AAC and AAT. Tyrosine also has two- TAC and TAT. This new strain is therefore due to A to T transversion at the first position of the putative codon. 

When a purine is replaced by a purine or pyrimidine by a pyrimidine, it is called a transition. Crossovers between purine and pyrimidine is called transversion. In general mutations caused by transition outnumber those due to transversions manyfold. The commonest mutations are due to C to T transition as cytosine bases are prone to be methylated at the 5'position and thence are spontaneously deaminated to thymine. Thus, the WHO database for COVID 19 in Feb 2020 showed 1670 C to T transitions compared with only 128 A to T transversions.

Thus, it is fair to say that the N501Y mutation in the VUI-202012/01 has persisted because it offers a survival advantage- i.e. infectivity, much as the G614 variant was more infective than the D614 variant. However, it must be said that there is no evidence that it is more dangerous. If anything, the D614G carried a lower mortality than the original strain, although this may have been due to better established treatment protocols.

Nor is there any evidence to suggest that the current vaccines will be less effective against the new strain. The current vaccines were in fact formulated against the original COVID strain and are just as effective against the D614G strain. This is due to the fact that protective antibodies target several epitopes and unless there is a significant conformational change in the shape of a protein, the vaccine generated antibodies will still neutralise.

I do think that this particular variant will take over despite the restrictions- just as D614G did. I also think there need be no undue cause for concern. With rapidly executed vaccination programmes, it should be possible to control the virus by spring.

Getting The Most Out of Azathioprine

 Azathioprine is widely used in Rheumatology for conditions such as vasculitis and Lupus, and by some in Rheumatoid. It certainly has a place in gastroenterology for the management of ulcerative colitis and Crohn's disease, where it is more effective for maintenance of remission than 5-ASA.

However, the use of Azathioprine is beset with problems. Around 10% of subjects have hypersensitivity reactions to this drug, comprising fever, nausea and diarrhoea, fatigue, malaise and myalgia, mandating rapid discontinuation. A further 25-30% have side effects such as hepatotoxicity in the form of transaminitis or myelotoxicity manifesting as neutropenia. As a result, fully 40% of subjects that start azathioprine do not continue with the drug.

Observance of some simple principles can obviate these difficulties. The first of these is widely practiced- measuring TPMT levels before starting azathioprine. In the general population, 89% of subjects are homozygous for high metabolism of azathioprine (high TPMT) , 11% are heterozygous and 0.3% have low levels of TPMT. 

It is common practice not to use azathioprine in subjects with lowish TPMT- these are the heterozygotes. This is a missed opportunity as these are the very subjects who are likely to respond the best to the drug. Thus, people with TPMT levels >25 U/ml need no dose adjustment. (Below 25 U/ml, halve the dose, and monitor more frequently). Conversely, those with levels above 65 U/ml, although reassuring on the face of it, are likely to have treatment failure.

Azathioprine is a prodrug of 6-MP. Most of the ingested azathioprine is non-enzymatically cleaved to 6-MP in the liver. Yet, there is a marked non-familiarity with 6-MP amongst Rheumatologists and perhaps to a lesser extent among Gastroenterologists. Several studies show that in subjects with hypersensitivity to azathioprine, nearly 70% tolerate a switch to 6-MP. The dose for 6-MP is half that of azathioprine (1-1.5 mg/kg body weight, rather than 2-2.5 mg/kg). Subjects with flu like reactions, nausea, emesis, myalgias and arthralgias on Azathioprine are likely to be able to switch successfully to 6-MP. OTOH, those with hepatotoxicity and pancreatitis are likely to have the same problems with 6-MP.

The converse does not apply. Thus, subjects who are intolerant to 6-MP should not be switched to azathioprine.

While most practitioners are aware of the importance of measuring TPMT before commencing thiopurines (azathioprine or 6-MP) , there is less awareness of the rather high usefulness of measuring thiopurine metabolites. Two metabolites are measured by most labs- 6-Thioguanine nucleotide (6-TGN) and 6-Methyl mercaptopurine (6-MMPN). The metabolite 6-MMPN is produced en-route to 6-TGN (please see diagram). It is the blood level of 6-TGN that indicates efficacy of azathioprine. Within a blood 6-TGN range of 235-450 pmol/8 x 10^8 RBC (send whole blood in an EDTA tube, just like TPMT), azathioprine is likely to be effective. Below 235, efficacy is likely to be low. This latter could be due to 2 reasons- non compliance, or the fact that not enough azathioprine is being metabolised to the active metabolite 6-TGN. In the latter case , blood 6-MMPN levels will be high (range 0-5700). 

The combination of low 6-TGN and high 6-MMPN levels presages treatment failure and hepatotoxicity, and is described as azathioprine resistance. Unlike with non-compliance (where 6-TGN is low and 6-MMPN is normal), increasing the dose of azathioprine where resistance exists is only likely to lead to hepatotoxicity without increasing efficacy.

Keep in mind though that unlike hepatotoxicity, isolated neutropenia is a surrogate marker for the effectiveness of azathioprine. Here, the 6-TGN levels are likely to be high, and may require a reduction in dosage, rather than discontinuation, as with transaminitis. (If hepatotoxicity and neutropenia occur together, discontinue the drug).

There are anecdotal reports that in subjects with high 6-MMPN levels, splitting the dose of azathioprine is likely to improve efficacy and reduce toxicity, while lowering the level of 6-MMPN and maintaining that of 6-TGN. However, this is based on observations from a single study.

It is fair to say that we could be using azathioprine a lot more effectively than we currently do.

 Figure. Thiopurine metabolic pathway. Metabolic pathway for AZA and 6MP is shown in the diagram. AZA: Azathioprine; 6-MP: 6-mercaptopurine; 6-TU: Thiouric acid; 6-MMP: 6-methylmercaptopurine; TIMT: Thiopurine methyl-transferase; 6-MMPR: Methyl-mercaptopurine ribonucleotide; TXMP: 6-thioxanthosine monophosphate; 6-TGN: Thioguanine nucleotide; 6-TG: Thioguanine; 6-TGDP: 6-thioguanine diphosphate; 6-TGTP: 6-thioguanine triphosphate; XO: Xanthine oxidase; TPMT: Thiopurine methyltransferase; HPRT: Hypoxanthine phosphoribosyl transferase.

Reference:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208360/

The Purinergic Pathway in Inflammation, Heart Disease & Cancer

 So you drink a cup of coffee because you are tired. It relieves your fatigue and headache? How does it do it? 

Or for that matter, how do methotexate and sulfasalazine relieve inflammation in rheumatoid arthritis or inflammatory bowel disease? How do clopidogrel and dipyridamole work? And what are the most important cellular markers of effective adoptive cell transfer therapy?

The answer to all these questions lies in the all-important purinergic system. This is the dance between the nucleotides ATP and ADP on one hand and adenosine on the other. They often have diametrically opposite effects in health and disease.

ATP is of course the currency of energy in eukaryotic cells. However, here we are referring to extracellular ATP, acting in a paracrine fashion on specific receptors. Such extracellular ATP may be released from necrotic cells, or leave apoptotic cells through pannexin channels or exit inflammatory cells such as neutrophils through connexin channels- connexin 37 & 44, specifically. They can also be part of vesicles, released from cells through exocytosis.

Whatever the origin of ATP, or the closely related dinucleotide ADP, formed from ATP, they act through two groups of cell surface receptors- the first, called P2Y receptors, are metabotropic receptors, i.e. they directly respond to the nucleotides- these are G-protein coupled receptors, and the second, called P2X receptors, are ionotropic receptors, i.e. ligand gated ion channels that open in response to inward flux of calcium (mostly) or sodium, or outward flux of potassium.

On the other side of the spectrum, sit P1 receptors, which ligate extracellular adenosine. These are also GPCRs. There are 4 types, adenosine receptor A1 (also called ADORA1), A2A(ADORA2A), A2B(ADORA2B) and A3(ADORA3). Of these, A2A and A2B are coupled to Gs and lead to increased levels of cellular cAMP, resulting in profound immunosuppression. A1 & A3 inhibit the formation of cAMP through Gi/o and are therefore generally immune promoting. While A1, A2A and A3 are high affinity adenosine receptors, A2B has low affinity, and is only stimulated under pathologic conditions such as high prevailing levels of adenosine in a hypoxic tumour microenvironment.

How is adenosine formed extracellularly? It is principally formed by the action of 2 sequential cell surface enzymes called CD39 and CD73. CD39 is an ectonucleoside triphosphate diphosphohydrolase, which converts ATP and ADP to AMP. CD73 is an ecto-5'-nuleotidase, that converts AMP to adenosine.

Adenosine can sometimes be generated by other enzymes from ATP & AMP, namely alkaline phosphatase, which has been described as a "promiscuous" enzyme.

Extracellular adenosine is short lived and pushed inside the cell through a couple of channels called Equilibrative Nucleoside Transporters 1&2 , also called ENT1 & ENT2.

Extracellular ATP is pro-inflammatory. It activates the NLRPR3 inflammasome in neutrophils and monocytes. The resulting Caspase1 cleaves pro-IL1 and pro-IL18 into their active forms. 

Adenosine, on the other hand is anti-inflammatory. It reduces inflammation through the A2A receptors present on neutrophils and lymphocytes, by increasing levels of cAMP. Remember, A2A is a high affinity receptor for adenosine.

Methotrexate and sulfasalazine owe their anti-inflammatory effect at least partly due to the fact that they stimulate CD73, which increases the formation of extracellular adenosine from AMP.

What of the A1, A2B and A3 receptors for adenosine? These have all been exploited pharmacologically. The heart blocking effect of adenosine in terminating SVT is exerted through the A1 receptor, while its vasodilating effect in cardiac stress testing is due to its action on A2B receptor on vascular endothelial cells. Stimulation of A3 receptors in non-pigmented cells in the anterior chamber of the eye leads to increased production of aqueous humour, and can be useful for treating sicca symptoms.

Dipyridamole inhibits the ENT1 & 2 channels, thus leading to increased levels of  extracellular adenosine. Hence its use in pharmacological cardiac stress testing.

As adenosine is formed from ATP and ADP, their levels vary inversely with each other. Thus, in inflammatory bowel disease, tissue hypoxia leads to increased production of HIF, which, acting as a transcription factor, stabilises the promoters for CD73 and A2B. A similar transcription factor called Sp1 binds to and stabilises the promoter for CD39. The net result is an increase in extracellular adenosine and a reduction in ATP. This leads to reduction in inflammation, both due to a fall in extracellular ATP levels and stimulation of A2A receptors on neutrophils and lymphocytes by adenosine. The latter also stimulates A2B receptors and maintains epithelial integrity, presumably by promoting healing through augmented blood flow.

The purinergic system, in particular ADP, plays an important role in the function of platelets. Thus, ADP stimulates P2Y1 receptors on platelets and through the G-protein Gq, activates phospholipase C. The downstream effect of this is change in the shape of platelets through the actin cytoskeleton. Similarly ADP activates the P2Y12 receptor, which, through the intermediation of the Gi G-protein switches off adenylyl cyclase, decreases cAMP and activates the GPIIa/IIIb receptor, thus facilitating the binding of platelets to fibrinogen, resulting in platelet aggregation.

Clopidogrel is a P2Y12 inhibitor. It is a prodrug and needs to be activated in the liver. This particular stage can be affected in certain mutations and thus reduce the efficacy of clopidogrel in affected subjects.

Stimulation of A2A and A2B receptors leads to platelet inhibition, explaining the efficacy of dipyridamole in prevention of ischaemic stroke.

While extracellular adenosine is regarded as a safety signal in ischaemia and reperfusion, where it reduces inflammation and tissue damage, it can have the opposite effect in cancer. In general, while its immunosuppressive effect on T-lymphocytes reduces autoimmunity, it can be a hindrance in fighting cancerous cells. In a recent paper in Nature, the investigators found that the subset of cancer sufferers who had the highest benefit from adoptive T cell therapy had a higher proportion of CD8+CD39-CD69- T-cells in the infusate. It is possible that increased expression of CD39 on T-cells leads to production of extracellular adenosine, immunosuppression and T-cell exhaustion, although this is yet to be confirmed. In general, adenosine is found in higher quantities in hypoxic tumour microenvironments, although this may be consequence rather than cause of tumour survival.

Finally, to the salutary effects of that cup of coffee. It is thought that caffeine reduces fatigue by inhibiting cerebral adenosine A2A receptors.

The Science Behind DNA Analysis of Archaeological Human Remains

 DNA, even when well preserved, will not last for more than a few hundred thousand years. Therefore the description of DNA sequences in million year old dinosaur remains is due to contamination from modern ambient DNA, which is widespread in labs and elsewhere. The PCR will amplify any DNA, modern or prehistoric.

There are however certain clues to the age of DNA remains. The first is fragmentation. Ancient DNA fragments are between 50 and 500 bases long due to degradation. In fact most are no longer than 100 nucleotides.

Second, and most reliable is the observation that at both ends of DNA (5' and 3'), Cytosine (C) is deaminated to Uracil (U). This is an effect that occurs after death due to bacterial cytosine deaminase. This latter enzyme is not present in mammals (not to be confused with AID or activation induced cytidine deaminase, which is present during development and plays a major role in the affinity maturation and somatic hypermutation of B cells, in effect giving B cells their antigen specificity).

Thus if an archaeological remains has a high proportion of uracil bases in DNA- remember, this base is only present in RNA, it indicates that the specimen is thousands of years old. Archaeologists use an enzyme called uracil-DNA- glycosylase (UDG) to break the bond between deoxyribose sugar and uracil and remove the uracil bases. 

Not doing so leads to a curious phenomenon. The PCR used to amplify the DNA remains reads the uracil as thymine (T), since thymine is the native DNA base, not uracil. Hence, on the supplementary (anti-sense) strand, it puts an adenine in place of the guanine (which paired with the original cytosine). The uracil on the sense strand is now replaced with thymine to pair with adenine. The original C-G pairing has therefore now been replaced by an A-T pair. Thus there is miscoding caused by the PCR process manifesting as "transition " of C to T and G to A. (In contrast, when a purine is replaced by pyrimidine base, it is described as "transversion"). 

There can be blocking lesions caused by the formation of non-physiological chemical bonds between say DNA & protein, which can make it difficult for PCR to read through the ancient DNA. This is difficult to overcome.

Archaeologists have databases comprising thousands of single nucleotide polymorphisms (SNPs) mapped from certain lineages such as Armenian farmers or Iranian invaders which they then compare with the putative SNPs from the sequenced DNA sample to see if there is a match.

Here, mitochondrial DNA, which is of course maternal derived (the sperm does not contain mitochondria) is more useful than nuclear DNA in lineage tracing. Although mitochondrial DNA shares the same predisposition for degradation with time elapsed since death as nuclear DNA, there are far more copies of a given DNA sequence in mitochondria than in chromosomes. Thus, while the nucleus will only have two copies of a given allele (on the 2 chromosomes), each mitochondrion has 10-100 copies- a phenomenon known as heteroplasmy, which explains why mitochondrial mutations cause such variable phenotypes unlike mutations that occur in chromosomes. Mitochondrial DNA therefore is favoured by archaeologists for lineage tracing.

Are there any other ways to determine where a long dead person originated, apart from DNA sequencing? As it happens, there is.

The isotopes of Strontium which are deposited in bone and teeth (dentine and enamel) during development are a reflection of the area where the person grew up in. There are 4 isotopes of Strontium- Sr84, Sr86, Sr87 and Sr88. Sr 87 is not a natural isotope. It is formed by beta decay from Rubidium 87. The rocks and water and therefore, by extension, the remains of a person or animal growing up in a certain area will always display a specific Sr87:Sr88 ratio that is native to that area. When that person migrates to a different geographical area, his teeth, which are often well preserved thousands of years after death, will still carry the signature Sr87:Sr88 ratio of the area where he or she grew up in , as the signature is established in the developing teeth or bones, and does not change after death. 

It is thus possible to say that the woman's remains found in a Harappan valley, in fact grew up in Iran.

 

Paradoxical Effect of Caesin Kinase Inhibition in Deletion 5q Myelodysplastic Syndrome

 Caesin Kinase (CK1) is common to the Wnt-beta catenin pathway and the Hedgehog pathway. Along with other kinases, it acts as an inhibitor in both pathways to reduce the transcription of genes in response to wnt and hedgehog proteins respectively. 

Myelodysplastic syndrome (MDS) due to deletion of 5q (5q del) has an unique phenotype which can be explained by the differential action of CK1 on the wnt-beta catenin pathway. 

Subjects with 5q del have macrocytic anaemia, mild thrombocytosis with dysplastic, hypolobated megakaryocytes. MDS in 5q del is uniquely responsive to immunomodulators such as lenalidomide, although the anaemia does relapse after 2-3 years due to new mutations in other genes such as RUNX1.

Deletion of 5q leads to haploinsufficiency of the CK1A1 gene located on 5q.32. Lenalidomide inhibits the remaining CK1A1 allele and reverses the phenotype of MDS. This is admittedly non-intuitive, as there seems to be an abrogation of the dose-response effect here. If haploinsufficiency leads to a phenotype, how can suppressing the remaining normal allele reverse that phenotype (rather than worsen it)?

This apparent paradox is explained by the effect of CK1 on the wnt-beta catenin pathway. Haploinsufficiency of CK1 removes some of the inhibition exercised on the wnt-beta catenin pathway. The latter leads to enhanced survival and proliferation of the neoplastic clone, causing MDS.

However, genetic knockdown in mice or pharmacological inhibition of the remaining CK1A1 allele by lenalidomide in 5q del MDS sufferers leads to complete disinhibition of the wnt-beta catenin pathway. While this initially stimulates the haemopoetic progenitors of the neoplastic clone, continued stimulation soon leads to stem cell exhaustion and death of the progenitor cells now bereft of any CK1 activity.

Reversal of The Earth's Magnetic Axis

 The magnetic axis of celestial objects flips periodically, including a complete reversal (by 180 degrees). For example, the sun's magnetic axis reverses direction every 11 years. However, the Earth's geomagnetic axis flips much less often. The last time it reversed completely was 780,000 years ago, a phenomenon called the Matuyama-Bunhes reversal after the two scientists who described it. Since then, the Earth's magnetic field has tried to flip on 10 occasions, but on each occasion it has reverted back to its current axis.

It's fair to say therefore that such reversals happen very infrequently for the Earth. When it does happen though, it affects the polarity of magnetic material in lava flows, sea beds etc which can be detected in rocks and fossils. 

The Curie temperature is one above which magnetic substances (which includes all minerals containing iron, nickel and cobalt) lose their magnetic properties. This varies between 580 and 680 degrees Celsius for the oxides of iron -Fe2O3 and Fe3O4. Conversely, when cooled below this temperature, such objects regain their magnetism. Thus, igneous rocks have inherent magnetism dating back to the time when they were formed from cooling lava flows millions of years ago.

The reversal of Earth's magnetic axis is not instantaneous- it occurs slowly- over thousands of years. During this period, magnetic material in cooling magma will take up the polarity of the reversed magnetic polarity of Earth, and the rocks formed therefrom would reflect this reversed polarity for ever. Thus, geologists can analyse such rocks, or fossils which have enclosed magnetic material in sea beds and make a fair guess as to their age. This is one way of fossil or rock dating.