SARS-CoV2 (Continued)

SARS-CoV2 (Continued)  

     One of the puzzling features of this virus is that some infected people, while carrying a considerable viral load, and shedding infectious virions, nevertheless develop such trivial symptoms that they never notice they are infected. While other, of course, develop virally driven hyper-inflammation,  respiratory failure, and sometimes also kidney and heart failure.

     This variability in response is especially striking when it affects a whole nation-state. Thus the official WHO figures record that Vietnam, with a population of 97 million, has confirmed only 416 cases of COVID-19, and that none have died. (c.f. UK, population 67 million, 297,914 cases, 45,677 deaths on 24th July. )

Possible explanations for variable responses.

     The hypothesis of genetic variability in the human host, which supposes that the Vietnamese lack e.g. the ACE2 receptor site (See my SARS-CoV2 post), is more-or-less ruled out by anecdotal observations such as that of an asymptomatic carrier infecting 5 family members [1]. As also is the hypothesis of genetic variability among the circulating SARS-CoV2 strains, for the carrier would obviously infect the household with the strain she was carrying. 

     Could there be competition between two co-infecting viral strains, where one causes trivial, often negligible, symptoms but occupies all the binding sites? 

     Or could there be, in some people, residual anti-bodies at a sufficient titre from a previous infection by the same (or sufficiently similar) coronavirus?  This last seems the best hypothesis, and in the last 10 days has received some support. 

    The group of Antonio Bertoletti at the Duke-NUS Medical School in Singapore has just published in Nature [2] an online report showing that previous infection with a virus of the beta-coronavirus family can leave long-lasting and multispecific T cell immunity to the nucleocapsid structural protein (N protein, or NP, See my Coronavirus post) that can cross-react with the N protein of SARS-CoV2). This previous infection could be a harmless "common cold" member of the corona virus family, but in Singapore it was possible also to study survivors of the 2003 SARS pandemic. 

     Back in 2013 a group in Taiwan explored the antigenicity of the N protein of the mild common cold virus HCoV-OC43, and had found that the middle section was highly antigenic [3]. Well over 90% of healthy young adults contained antibodies in their serum against the N protein of this common virus. These antibodies were even found in cord-blood samples showing that newborn babies acquire some immunity against coronaviruses from their mothers. 

     We have already learnt that it is foolish to infect yourself deliberately with SARS-CoV2; you could become very ill or die. But there may be a beta-coronavirus, prevalent in Vietnam, that does protect you against COVID-19. And it may be that here in Britain a sufficiently recent 'common cold' may leave you with enough circulating antibodies to prevent or greatly limit the effect of SARS-CoV2 infection. 

References

[1]  Susan Lee,  Paula Meyler,  et al. (2020) Can J Anaesth. : 1–7. "Asymptomatic carriage and transmission of SARS-CoV-2: What do we know?". 

[2]  Le Bert N, Tan AT, Kunasegaran K, et al. (2020)  "SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls." [published online ahead of print]. Nature. 2020;10.1038/s41586-020-2550-z. doi:10.1038/s41586-020-2550-z.  

See also  Ruairi J. Mackenzie, Science Writer for Technology Networks (2020) “'Common Cold' Coronaviruses Could Help Produce Anti-SARS-CoV-2 Immune Cells." 

[3]  Fang-Ying Liang, Leng-Chieh Lin, (2013  J Virol Methods;187(2):413-20. "Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: implications for the diagnosis of coronavirus infection. "

COVID is not retreating

Easing the Lock-down-2

(COVID-19 is no longer retreating)

     Using governmental data  for the "new UK cases each day", the numbers have been declining markedly since I started logging them on the 1st of May.  Then we were getting 6000 new cases per day, now only 500 - 700. 
     Now, if there are fewer active cases in the community there should be fewer new cases; i.e. the decline should be exponential, or logarithmic, like a cooling curve.  So, in the Figure below I have plotted the logarithm of the daily increment in UK cases against days (since the 1st of May, so 85 is the 24th July). (A value of 3.0 would mean 1000 new cases per day (1000=10^3); a value of 2.0 means 100 new cases per day (100=10^2).) 
    The data from 1st May to the 24th June are coloured blue, and fit a logarithmic decline. However, the data from 30th June to the 24th July, coloured red, show that the disease is no longer on the decline. We are in steady-state. 
     At the worst there could be a "second-wave", as we have been warned; at the best we shall live like this indefinitely, or until there is an effective and safe vaccine.

Semi-log plot of new cases per day in UK

(See text for details)

SARS-CoV2

SARS-CoV2

    After looking briefly into coronaviruses in general (see previous post), I have turned to SARS-CoV2, the virus responsible for the current pandemic of the respiratory infection called COVID-19.

Interesting aspect at this stage in the pandemic include:

• Important biochemical and biological differences between SARS-CoV2 and SARS-CoV viruses.
• Hight infectivity; 100 or 1000 times higher than SARS-CoV.
• Why are 30-40% of infected  'carriers' symptom-free?

Difference between SARS-CoV2 and SARS-CoV(1) that might cause higher infectivity.

    SARS-CoV2 is said to be 1000 time more infectious than SARS-CoV1; a pretty loose statement, but there is some biochemistry to investigate. Is this high infectivity due to:  

a.    different, more accessible or numerous, targets on host;

b.    tighter binding to target; 

c.     epidemiological factors like shedding before or without symptoms, or more coughing and sneezing; 

d.    better evasion of host responses. 

[a] Target on host

    The host receptor for both SARS-CoV and SARS-CoV2  seems to be the dimeric membrane-bound protease called ACE2 (for Angiotensin Converting Enzyme 2). There is a small mystery here, as the first investigators found very little ACE2 protein, or mRNA, in human lung tissue, though lots in arteries, gut, kidney, testes and elsewhere [1]. Yet SARS-CoV2  seems to attack the lower respiratory tract (as well as gut, blood-cells, kidney, etc). This was so important that the question was re-examined and some ACE2 was found in lung tissue, particularly around arterioles [2].  (It is notable, however, that SARS-CoV2  can cause diarrhoea and kidney damage [3].) Other coronaviruses act primarily as gut pathogens (See previous blog). I worried that the polyclonal antibody used by Hamming to test the presence of ACE2 (which was reared against a stretch of 19 amino acids distinctive to ACE2) might cross-react and mislead. However, it seems to be universally accepted that the receptor for SARS-CoV2  is ACE2.

[b] Tighter binding of Spike to Target.

    The spike protein is not highly conserved; the opposite rather, and it seems likely that mutations, deletions and insertions in spike can affect host range; possibly infectivity as well. There are distinctive features in the spike protein of SARS-CoV2 not found in SARS-CoV spike.  Thus, there is an insert of 4 amino acids (PRRA) into the sequence, which generates a cleavage site absent from the spike protein of SARS-CoV and several other coronaviruses (but present in MERS!). 
                                                                                ↓            
            SARS-CoV2: CASYQTQTNSPRRARSVASQSI
            SARS-CoV  : CASYQTQTNS­­– – – –RSVASQSI
Cleavage is effected by a host protease present cytoplasmically throughout the body. It is a 'subtilisin-type' calcium-dependent protease (called furin), which cleaves after the marked serine residue, but the cleavage site is flagged by the paired basic amino acids (–R+R+–). The furin enzyme is obviously present to service host proteins. But SARS-CoV2 is not unique among viruses in using it for pathologicial purposes, for furin also operates in the activation of: HIV, influenza, dengue fever, Marburg virus, papillomavirus, and even anthrax toxin. It is suggested that processing of progeny virions before release may facilitate the spread of virus (c.f. SARS-CoV)[4]. 
    The spike protein of SARS-CoV2 operates as two peptides (S1 and S2) formed into a trimeric "clove-like" structure.  Tai et al. (2020) were able to compare the binding (to human ACE2) of SARS-CoV2 spike with that of SARS-CoV spike, and found it bound 9 time more tightly. (Interestingly, it bound even more tightly to bat ACE2). [5] 

[c] Epidemiological factors.

     It is important to distinguish pre-symptomatic from truly a-symptomatic carriers; both categories of infected subjects experience no symptoms, but in the former case they eventually develop symptoms, while in the latter they never do. When I use the term 'asymtomatic' in this post it will always mean that the subject did not develop sysmptoms, in at least 4 weeks. Both categories can spread the disease.
    Compared with SARS-CoV of 2003, SARS-CoV2 causes more cases with mild (or very mild) symptoms, and larger numbers stayed at home in the community. There was also twice as long incubation period before the appearance of symptoms (4–12 days). Similarly relevant for the spread of the disease, the new strain can shed infective particles as soon as symptoms appear; or even before (see above). And they can continue shedding for 3 weeks [6].   Susan Lee et al. [7] mentions a family in Anyang (China) where an asymptomatic carrier tested positive for the virus and infected 5 family members. 
    Other factors of obvious relevance to infectivity are propensity to cough or sneeze. 

[d] Evasion of host defences.

    Two recent well referenced summaries are by Indwiani Astuti and Ysrafil [8] and Swatantra Kumar et al. [9]

 References

[1] Donoghue, M., Hsieh, F. et al. (2000) Circulation Research. 87:e1–e9; "A Novel Angiotensin-Converting Enzyme–Related Carboxypeptidase (ACE2) Converts Angiotensin I to Angiotensin 1-9".
[2] Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H.  (2004)"Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis." J Pathol.; 203:631-637. doi:10.1002/path.1570
[3] Martinez-Rojas, M.A. et al. (2020) "Is the kidney a target of SARS-CoV-2?"; Am J Physiol Renal Physiol.; 318:F1454-F1462.
[4] Coutard, B., Valle, C. et al  (2020)
Antiviral Res.; 176: 104742. "The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade."
[5] Tai, W-B. He L.,  Zhang, X-J. et.al. (2020) Cellular & Molecular Immunology volume 17, 613–620. "Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine"
[6] Petersen, E., Koopmans, M. et al (2020).Lancet, Infectious Diseases, https://doi.org/10.1016/ S1473-3099(20)30484-9 "Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics.".
[7] Lee S, Meyler P, Mozel M, Tauh T, Merchant R. "Asymptomatic carriage and transmission of SARS-CoV-2: What do we know?"  Can J Anaesth. 2020;1-7. doi:10.1007/s12630-020-01729-x
[8]  Indwiani Astuti & Ysrafil, (2020) Diabetes Metab Syndr. 2020 July-August; 14(4): 407–412.
Published online 2020 Apr 18. doi: 10.1016/j.dsx.2020.04.020 "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response"
[9]  Kumar S., Nyodu R., Maurya V.K., Saxena S.K. (2020) Host Immune Response and Immunobiology of Human SARS-CoV-2 Infection. In: Saxena S. (eds) Coronavirus Disease 2019 (COVID-19). Medical Virology: From Pathogenesis to Disease Control. Springer, Singapore. Published online 2020 Apr 30. doi: 10.1007/978-981-15-4814-7_5

Corona Viruses

Corona Viruses

(Some notes on Coronaviridae to put SARS-CoV2 into context)

Coronaviridae – the Corona Virus family

     The name “corona virus”, and the biological family name Coronaviridae, were coined in 1975 by Tyrrell and co-workers. However the viruses had been discovered ten years earlier, in 1965 by Tyrrell et al., and independently (in 1966) by Hamre & Procknow, in tissue or organ cultures inoculated with material from volunteers with “common colds” [1]. The salient features at this stage were [a] RNA viruses, [b] ether-sensitive (so with a lipoid envelope), [c] cultivable in cultured human tissue or organ cultures but not in fertilised chicken’s eggs, [d] isolated from volunteers with mild upper-respiratory tract infections [1].

     With the electron microscope it was possible to visualise particles of 80 to 150 nm diameter (c. 10^-4 mm), decorated with widely spaced club-shaped knobs, or ‘spikes’; this picture has become well-known the world over during the pandemic of SARS-CoV2 of 2019/2020 [1].  (It has been suggested that the name refers more to a solar corona than directly to a crown or coronet.)

    Considerable progress was made between 1965 and 2002 in understanding the biology of the these ubiquitous viruses that caused mild disease symptoms. Especially studied were two strains called OC43 and 229E. The continuous RNA strand is approximately 30,000 bases long, single-stranded and is in the ‘positive’ orientation (so it can be directly translated into protein, 5’-terminus of RNA corresponding to NH2-terminus of protein).  Like other RNA viruses, the genome is susceptible to frequent mutations. Immunity of the host tends to be transient, so an endemic strain of virus (like 229E in the USA) can cause wave after wave of epidemic disease in a population, recurring every 2-3 years [1].

Taxonomy and Evolution

     It is now believed that corona viruses are very ancient in evolutionary terms, having been around some 300 million years, i.e. as long as their bat and bird hosts [2]. Identification and taxonomy are both based on cloning up complimentary DNA using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), employing primers synthesised to match highly conserved parts of the genome; often in the RNA-dependent RNA polymerase (RdRP), coded at the overlap of the two large open reading frames (See Figure below). There are now held to be 4 genera of viruses in the family Coronaviridae (see Table). 

Table

Figure [See ref. 3]

    With the outbreak of Severe Acute Respiratory Syndrome (SARS) in China in 2003, interest in the Coronanviridae intensified. This was a dangerous disease with average fatality of those infected of around 10%, and much higher for the elderly. It spread readily from human to human by droplet infection, and indirectly by touching contaminated surfaces. It was found to be caused by a new coronavirus, named SARS-CoV. This strain is related to bat viruses, but seems to have jumped to a human from a palm civet [3]. Cases were eventually reported in 29 countries, mostly with links to east Asia. But the disease then died out. Apart from a small escape-infection from a Chinese laboratory in 2004 it has not been seen since, anywhere in the world. 

    In 2013 another novel coronavirus disease broke out in the Middle East with an even higher mortality rate of 35%. This Middle East Respiratory Syndrome (MERS) has remained confined to the Middle East, where it lingers still (some 14 cases were reported in the first 5 months of 2020). While SARS-CoV seems to have jumped from bat to man via civet cats, the MERS virus seems to have jumped via the dromedary camel, which may explain its restriction to Saudi Arabia and neighbouring countries.  It has been endemic in camels for at least 3 decades [3].

    Over the last 17 years, intense study of the SARS-CoV virus  (which for clarity I shall sometimes call SARS-CoV1) has laid the foundation of our knowledge of SARS CoV2, the closely related agent of our present COVID-19 pandemic. I shall therefore expand a bit on the biology of SARS-CoV1.

SARS CoV1 Molecular Biology

     This, like all coronaviruses, is an enveloped, RNA virus containing a single-stranded, positively orientated, RNA molecule of 29,751 nucleotides [4]. This RNA codes for 28 proteins: 4 Structural proteins (S=spike protein, E=envelope protein, M=membrane protein, N=nucleocapsid protein); 16 non-structural proteins, derived by cleavage from two large polyproteins; and 8 accessory proteins, so-called because they are non-essential in tissue culture, though presumably important in the wild. The 2 polyproteins are coded by Open Reading Frame 1a (pp1a) and Open Reading Frame 1b (pp1b). 

    The functions of S, M, and N are relatively straightforward; not so the multi-functional E protein:

● S, a glycoprotein expressed on the outer surface of the excreted virion, is the docking mechanism of the virus, and contains specificity for the host target site. In the case of SARS-CoV1 the target is human Angiotensin-Converting-Enzyme 2 (ACE2), which is primarily expressed in the lower respiratory tract. (The HCoV-229E S-protein is specific for the host protein CD13; the MERS-CoV spike binds to dipeptidyl piptidase, so target gut and kidney.) 

● M, the most abundant protein, and the one that defines the shape of the virion.

● N, a protein that binds the genomic RNA, (I suppose like the histones that wrap and protect our DNA). 

● E, the 'envelope' protein, contain a hydrophobic domain of 24-28 amino acids, so presumably spans the lipid membrane. It is the smallest of the structural proteins at 76 amino acids, binds to M in the virus membrane, where it may act as an ion-channel or 'viroporin' [5,6]. But a great fraction of the completed E peptides are not found in the membrane, and E seems to have several other functions. There is a PDZ-bininding-Motif (PBM) at the extreme carboxyl end of E, which suggests that it can bind to a PDZ motif on some host proteins. (Of the 320 such PDZ-containing proteins in the human, only 5 are known to bind the E protein of SARS-CoV1; (a) Na/K ATPase alpha-1 subunit, (b) stomatin, (c) syntenin, (d) PALS, and (e) BcL-xL. It is therefore easy to imagine that the virus can, by means of these protein-protein interactions, disrupt tight-junctions in the lung, and Na⁺ concentration in nerve and muscle, while (with syntenin) it could cause a 'cytokine storm'. [5] (See Pathology below.)

    Several of the non-structural proteins (nsp) are highly conserved. Thus, nsp1, coded at the extreme 5' end of the genome, is highly conserved between all coronaviruses, but has very little homology with anything else in protein and nucleotide data bases; it is unique to Coronaviridae. The protein, of about 20kD, may inhibit cell protein synthesis, perhaps by degrading messenger RNA.[7].  

    The RNA-dependent RNA polymerase (RdRP), coded at the junction betwee the two large open reading frames (see Figure) [8], is the enzyme that, with auxiliary proteins, replicates the genome, and creates smaller fragments of RNA that act as templates for protein synthesis [8]. RdRP is one of the most tightly conserved regions of the geneome and the primers used for diagnosis and taxonomy are usually based on sequence from this region. 

Pathology

A high proportions of cases of SARS-CoV1 (and especially of MERS) occurred among health care workers rather than close family members, from which it is can be inferred that shedding of infectious virions occurs well after the onset of symptoms and hospitalization. It was often found that, as symptoms of distress increase, viral load decreases. This suggested that part of the pathology is due to the immune response. (See also the 'cytokine storm' in Swine Flu.) A comparison between those that survive and those that succumb to severe infection points to a failure (in the latter) to switch from innate immunity to acquired immunity. [3] 

References

[1]  Kahn, Jeffrey S. &  McIntosh, K. (2005) The Pediatric Infectious Disease Journal: Vol 24, S223-S227, "History and Recent Advances in Coronavirus Discovery."

[2]  Wertheim J.O., Chu D.K.W., Peiris J.S.M., Pond S.L.K., Poon L.L.M. (2013) J Virol. 87, 7039–7045. "A case for the ancient origin of coronaviruses."

[3]  de Wit, E., van Doremalen, N., Falzarano, D., Vincent JM. (2016) Nat Rev Microbiol. ; 14: 523–534. 

[4]  Marco A., Marra, et al., (2003) Science, 300, 1399-1404

"The Genome Sequence of the SARS-Associated Coronavirus".

[5]  Wu, QingFa,  Zhang, YiLin, et al. (2003) Genomics, Proteomics & Bioinformatics, Volume 1, 131-144. "The E Protein Is a Multifunctional Membrane Protein of SARS-CoV".

[6]  Castaño-Rodriguez C, Honrubia JM, et al. (2018). mBio. 22;9(3):e02325-17.  "Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis."

[7]  Connor,  R.F. & Roper, R.L. (2007) Trends Microbiol; 15: 51–53. "Unique SARS-CoV protein nsp1: bioinformatics, biochemistry and potential effects on virulence."

[8]  Pasternak, A. O., Spaan, W.J.M., Snijder, E.J. (2006) J Gen Virol  87,1403–1421, "Nidovirus transcription: how to make sense…?"

Adrenergic Angina Pectoris

Introduction

     Angina pectoris is a pain in the chest, usually attendant on exercise. It is widely thought that stable Angina pectoris is a result of Ischaemic heart disease [1]. For this there are said to be a number of 'risk' or pre-disposing factors [2]:

• increasing age, 
• smoking, 
• high-fat diet, 
• lack of exercise, 
• overweight, 
• excessive alcohol consumption, 
• (other conditions, including high blood pressure, high cholesterol and diabetes, a family history of CVD, being of south Asian, African or African-Caribbean descent.)

     I shall assume that the rational man will neither smoke nor eat nor drink too much. Clearly, you cannot do anything about your increasing age, or your ethnic or familial inheritance. But I think it is highly interesting to know more about how all these factors impinge on the heart and its coronary blood supply (i.e. oxygen supply.)
    In my own case I was able to get my blood pressure and cholesterol values into the 'healthy' range by taking a low does of a statin for a few months (supplemented with CoQ to offset one of the deleterious effects of inhibiting HMGCoA synthase. [See my blog post]), and by eschewing completely the double-cream I liked to pour over my dessert. In addition I tweaked my consumption of alcohol down by 20%, and exercise up by 20% (aiming at ≧60 minutes walking per day, and running up and down stairs when a trip was required). As for 'long acting nitrates', after reading up the literature I never touched the packet of tablets I had been prescribed,  for it was clear that they cause desensitisation, and gratuitously mess with your glutathione (and all -SH groups in general). I used to carry a little vial of 'short acting' glyceryl trinitrate (GTN) tablets [see blog post 'Angina and GTN'] (as my father did, to his dying day aged 86); but I now find I can leave them behind without regret. The 'low dose aspirin' seemed counter-indicated in my case as there is no evidence of clotting [3]. 
     That I was prescribed a synthetic β1-blocker (Bisoprolol) interested me. What does adrenaline or noradrenaline have to do with blood flow?  They are the "fight or flight" hormones after all? As I am a minimalist when it comes to interventions, and because I had discontinued all the other pills, I first asked the doctor to halve the dose then quartered it by snapping the tiny pills in half, then stopped altogether my daily β-blocker. I was intent on lowering my adrenergic stimulation by more natural means, if at all possible.
      On the other hand, I had clear evidence that mental stress could cause me the ischaemic pain of angina in the absence of increased exercise-related oxygen demand. Resentment, aprehension, and fear all caused ischaemic chest pain even when I was physically at rest. Once, when playing the fiddle in a pub I realised that I had forgotten the second half of the tune. Suddenly, the familiar tightness in the chest; and I had to stop playing and sit down. Of course:  "the fight-or-flight response"! So, I have to look into mental stress as a cause (or even the cause) of my angina; for I can sometimes walk energetically without trouble from my heart.
     First, let us note that there are two rather similar adrenergic chemicals, adrenaline and noradrenaline; the former released into the bloodstream by the adrenal glands (and thus a 'hormone'), the latter released by nerves (and thus a 'neurotransmitter'). Both are catecholamines, both are referred to as 'adrenergic', and their activieties greatly overlap. 
    Next, let us note that there is an array of 9 (or more) closely related adrenergic receptors, all being 7-helix transmembrane proteins of the rhodopsin family, and GTP-binding-protein-linked receptors. They were divided into two basic types by Ahlquist in 1948 on the basis of affinity (alpha and beta). However, since then, a combination of gene sequencing and pharmacological analysis in transfected cells has led to the distinction of three main types: α1, α2, and β, with the identification  of  three  subtypes for each of them [4]. See table.

TABLE

Name Action Typical site

α1A       ↑Ca2+Ch artery
α1B        IP3/DAG heart
α1C        IP3/DAG (Not human)
α2A       ↓cAMP, ↑K+Ch,↓Ca2+Ch platlet, brain
α2B       ↓cAMP,↓Ca2+Ch
α2C       ↓cAMP, spleen
β1       ↑cAMP heart
β2       ↑cAMP lung
β3       ↑cAMP fat

Experiments using Endogenous Catecholamines and Pharmacological Agents.

     (1) Plunging the hand and wrist into icy water for 90 s (the 'cold pressor test') causes release of both adrenaline and noradrenaline. In healthy subjects, blood pressure rises markedly during immersion, but falls as steadily afterwards.   [5]
    (2)  In similar experiments, Monahan et al. found that, besides the increase in blood pressure, the catecholamines released by the icy-water caused pronounced vasodilatation in healthy young men (to supply the extra oxygen needed by the heart muscle), but that this effect was lost with age. Moreover, in young men, blocking α- and β-adrenergic responses mimics the effect of ageing, suggesting a critical role for an adrenergic mechanism in this ageing effect. [6] 
    (3) Nabel et al. (1988) applied the cold pressor test to healthy controls and patients with varying degrees of atherotic damage; group I healthy, group II mildly sclerotic with some smooth vessels and some damaged vessels, group III heavily sclerotic.  The catecholamine release produced vasodilation in group I. In group II the smooth segments dilated, while the damaged segments contracted. In group III only 2 smooth segments were found, but they dilated. The authors speculate that this constricting effect may represent 'altered catecholamine sensitivity' and/or a defect in 'endothelial vasodilator function'. [7]. 
    (4)  Heusch et al. (2000) review a number of indication that there is α-adrenergic coronary constriction whether evoked by cold water, exercise or emotional stress (a public speaking ordeal). Such α-adrenergic coronary constriction, augmented  by coronary endothelial dysfunction and atherosclerosis, is "powerful enough to induce myocardial ischaemia and limit myocardial function". [8]. 
    (5)  Recent studies indicate a genetic determination of α2-adrenergic coronary constriction. Apparently, a C to T polymorphism at nucleotide 825 in the gene coding for the Gβ3 subunit of G protein leads to shortening the protein by 41 amino acids and is associated with enhanced signal transduction, and hypertension, and also with pronounced sensitivity to α2-adrenergic coronary constriction.[9] Such individuals comprise 30% in caucasian populations [10].
   (6)  Experimentally, it is found that β-blockers blunt or ameliorate the pain of stable angina in 70% of cases. But not 100%.  [11]

Conclusions

     It is tempting to think that one's inherited genotype is at least partly to blame for one's angina, and that it is not entirely due to idleness and over-eating. My father had very similar angina at a similar age, and his mother also; both were lean and active people. 
     I am familiar with the autonomic nervous system going wonky in older people [12], such as sluggishness in the auto-regulation of cerebral blood pressure, and the the intense desire to urinate that leaves you as soon as you start walking. However, it seems possible that, when it comes to adrenergic effects on cardiovascular function, ageing might always mean 'sclerotic damage to endothelium', when you have the means to look for it. 
     Note [my post] that the endothelium seems to be the site of NO release from endogenous arginine, and that 'Heusch et al invoked an 'endothelial vasodilator function'. Is it the endogenous production of NO that is damaged in atherosclerosis?
     Note also [my post] that GTN seems to work by cGMP rather than cAMP, It is also the case that NO from GTN can (usually) relax coronaries even when catecholamines cease to cause coronary dilation and cause only contraction?  

References:

1. https://www.heart.org/en/health-topics/heart-attack/angina-chest-pain/angina-pectoris-stable-angina 
2. https://www.nhs.uk/conditions/atherosclerosis/
3. https://www.nhs.uk/news/medication/aspirin-bleeding-risk-balances-out-lower-heart-attack-risk-worried-well/
4. Strosberg, A.D. (1993) Protein Science, 2, 1198-1209.
5. Silverthorn, DU, and J. Michael (2013) "Cold stress and the cold pressor test"; Advances in Physiology Education Vol. 37, 93-96.
6. Monahan, KD., RP Feehan, et al. (2013) J Physiol. 2013; 591: 2937–2947.
7. Nabel EG, Ganz P, et al. (1988) Circulation, 77, 43-52
8. Heusch, G. et al. Circulation. 2000;101:689–694
9. Circ Res..1999; 85:965–969.
10. C. K. Naber, R. Erbel, W. Siffert.  Current Genomics (2003) Vol. 4, 337-342.
11. Elliott, WC., JM. Stone, (1969) Progress in Cardiovascular Diseases Vol. 12, 83-98.
12. Parashar, R., M. Amiret al. (2016) J Clin Diagn Res. 2016: CC11–CC15. "Age Related Changes in Autonomic Functions"

(Please comment direct to Cawstein@gmail.com)

Angina pectoris and Glyceryl Trinitrate

Introduction

     Angina pectoris is a pain in the chest, usually attendant on exercise. It is widely said that stable Angina pectoris is a result of Ischaemic heart disease [1], itself a result of excess ingestion of cholesterol, causing reduced flow of oxygenated blood in the coronary arteries (which form a 'crown-shaped' network round the heart).
     If you go to your doctor with chest pain in the UK (currently, i.e. 2017-2020) you will probably be investigated by a nurse (rather than a General Practitioner or cardiac surgeon). If you show curiosity about how bad your condition is, you will be told about various dangerous-sounding procedures that can examine how blocked your coronary arteries are, and where the blockage lies.  If you suggest improving blood-flow by lowering your intake of cholersterol the nurse pulls a long face, and you go away with a prescription for Statins (see Statins), and the impression that only a surgical reaming to scrape away the greasy plaques will restore your cardiac health. 
     However, because there are so many variables, it is very difficult for a nurse to say anything certain about your particular case, though she may be well versed in the average case. Your cholesterol may come down simply with diet. You may be among the 30% of caucasians who have a truncated form of the β subunit of the G-protein that confers hypersensitivity to adrenergic-constriction of coronary arteries [2]. Likewise, you may be a carrier of a polymorphism in the IL-6 gene. (It seems that patients carrying the 174C allele showed a 2-fold increased risk for angina pectoris (P = .036) over those carrying the 174G allele [3].)
     In August 2017 a specialist cardiac nurse confidently diagnosed my chest pain  as 'stable Angina pectoris' using a sloping treadmill while I was wired to an ecg-machine. My first clue that it was not exercise alone that caused my chest pain was my experience of what is called "walk through Angina" [4]; slowing my walking at the onset of pain I often found the pain lessening, even when I returned to my normal speed.  This observation is widely known in the literature, but inadequately explained. 
     Next, my interest was taken by the almost miraculous effect of the little pills containing 0.3 mg of glyceryl trinitrate (GTN), known since 1879 [5] to alleviate the exercise-induced chest pain known as stable angina pectoris. GTN was known to cause muscle relaxation, but its precise mode of action was not known for over a century; did it dilate the coronaries (allowing improved oxygenation of the ventricle), or did it dilate the systemic arteries (thus lessening the work-load and oxygen-requirement of the heart); or both? What did seem very unlikely is that GTN could unblock coronary arteries blocked by fatty plaques. And indeed, it is occasionally reported that GTN is without beneficial effect in such cases [6].
     It is now known that glyceryl trinitrate is broken down by enzymes in the body to release NO (the nitric oxide free-radical). Since 1986, we have learned that NO  is a wide-ranging physiological regulator of extraordinary simplicity, mobility, and short half-life [7]. It binds to the haem group in guanyl cyclase increasing its production of cyclic GMP (cGMP) with consequent smooth-muscle relaxation. GTN seems the perfect drug, as it is extremely cheap, fast acting in tiny doses and its effects seem more-or-less confined to this relief of ischaemic chest pain. With GTN pills in my pocket I did not feel my angina was a problem, and I began to wondered if stable angina is little more than a failure of the ageing body to produce sufficient endogenous NO. 

Mechanisms involved in the actions of nitrates

     After finding that NO relaxes smooth muscle, it was assumed that the main effect of NO on angina was in lowering the work that the ventricle had to perform against peripheral resistance [8], but it is now clear that coronary arteries are also relaxed [9]; so perhaps both more oxygen available, and less needed. 
    It seems that NO is largely generated (from arginine) by enzymes in the endothelium of blood vessels. It binds to a haem group of guanyl cyclase, stimulating that to produce more cyclic GMP (cGMP). That in turn stimulates protein kinase G (PKG) which phosphorylates (on serine or threonine residues) a number of protein targets depending on cell type. PKGs (of which there are several) are big players in the regulation of cytoplasmic calcium ion concentration (Ca2+), and it is here that the striking effects of GTN on the heart are evoked, for activated PKGs inhibit L-type Ca-channels and open Ca-dependent K+ channels, causing muscle relaxation. 
     Aspects of this NO-activated pathway parallel, and may overlap with, that of the the so-called adrenergic systems, i.e. the sympathetic nervous and hormonal systems that are known to cause large and rapid effects on blood flow.  Thus, there is evidence that catecholamines (adrenaline and noradrenaline) raise blood pressure by cAMP rather than cGMP [10]. Further aspects of the adrenergic control of blood flow will be considered in the next section [11].
    I end this section by concluding that NO from the tiny GTN pill, relaxes coronary and peripheral vessels by a mechanism involving cGMP and cytoplasmic Ca2+,  which is similar to, but not identical to, the mechanisms involved in adrenergic control. 

References:
[1] https://www.heart.org/en/health-topics/heart-attack/angina-chest-pain/angina-pectoris-stable-angina
[2] Circ Res..1999; 85:965–969.
[3]  Fernanda Amorim, Bianca P Campagnaro,  Clarissa L Tonini, Silvana S Meyrelles. Angiology 62:549-53 (2011) Association of Interleukin-6 Gene Polymorphism With Angina Pectoris.
[4] https://www.wikidoc.org/index.php/Walk_through_angina_pectoris
[5] William E Boden, Santosh K Padala, Katherine P Cabral, Ivo R Buschmann, and Mandeep S Sidhu; Drug Des Devel Ther. 2015; 9: 4793–4805.
[6] Bernstein, L., Friesinger, G. C., Lichtlen, P. R., and Ross, R. S. (1966). The effect of nitroglycerin on the systemic and coronary circulation in man and dogs. Myocardial blood flow measured with xenon133. Circulation, 33, 107.
[7] https://www.nobelprize.org/prizes/medicine/1998/summary/

[8] Kawakami H, Sumimoto T, Hamada M, Mukai M, Shigematsu Y, Matsuoka H, Abe M, Hiwada K.; Angiology. (1995), 46:151-6.; "Acute effect of glyceryl trinitrate on systolic blood pressure and other hemodynamic variables.".

[9] Nabel EG, Ganz P, et al. (1988) Circulation, 77, 43-52
[10] Strosberg, A.D. (1993) Protein Science, 2, 1198-1209., Circulation. (2018); 138: 1974–87.
[11] The next post in this blog.

The curve is not flattening.

COVID-19 Epidemiology, Part 6

    I last posted on COVID-19 epidemiology (from my perspective) on 15th April.  We are a month on and my optimism is draining away. Since then the doubling time in Northamptonshire rose encouragingly to a peak of 79 days, but has since slumped to 30, where it is like the average for the UK. The daily increment of cases in the UK, over the last 18 days, has fluctuated between 6000 and 3200 but stubbornly will not fall below 3200. 

    I keep asking a question that I do not see others asking — "Who are the new cases?"; and nor do I see any information from the government that will help answer it. Are they "front-liners", brave people who stand up and serve the public, facing hundreds of potential cases each day with only a pair of rubber gloves or a visor for protection; nurses, bus-drivers, cashiers? Or are they repatriated Britons from overseas? Or are they risk-takers, who have approached strangers more closely than 2 metres? The answers MUST be known and ARE important. 

    We want to know if 2 meters is enough distancing for protection; and the effectiveness of gloves and visors. We want information on the length of time between catching a virus, and showing symptoms; and during what part of that we are infectious. 

    Hanging above all that is a more general question —  we want to know if there is a prospect of relaxing our (self-imposed) distancing before the widespread availability of an effective vaccine in a year's time.