ICU resources for COVID-19, vitamin D and the immune system
Marik Protocol; Front
Line COVID-19 Critical Care Working Group Protocol; Farid Jalali's
beautiful detective work hypothesising about exactly what is happening
in the lungs of COVID-19 patients; Vitamin D to stabilize the
endothelium . . .
Robin Whittle firstname.lastname@example.org
. 2020-04-09 Last update 2021-11-23
Updated 2021-11-12 regarding the
first article below, Chauss et al. 2021, which is the peer-reviewed
successor to the preprint McGregor et al. 2020.
The updates/ page lists all the significant updates to these cv19 pages.
To the main COVID-19 page of this site: http://aminotheory.com/cv19/
, concerning nutritional supplements - especially vitamin D and boron -
for all adults and some children ASAP, to reduce or eliminate the
tendency for their immune system to be weak and/or dysregulated
(overly-aggressive, pro-inflammatory and so self-destructive) - which
is what causes severe symptoms with COVID-19. There you
will find multiple articles recommending that everyone should have at
least 40ng/ml (100nmol/l) 25OHD levels - and this starts with expectant
mothers and their newborns.
If everyone in the
world was already replete in vitamin D alone (with the current very
poor boron status) then I believe that far fewer people would be dying
or seriously harmed due to COVID-19. In the absence
of proper vitamin D levels, it is possible that boron repletion (such
as 6mg a day, instead of the usual 1mg or so) would be helpful
too. See the main page for more on boron and immunity.
Obesity ../obesity/ would still be a problem, as would some particular patterns of genes ../#haplotypes
- but the outcomes would be far less extreme than they are now, with
many or most of these people seriously deficient in vitamin D and
probably other nutrients. Please see the ../obesity/
for links to research concerning excess adipocytes expressing ACE2, releasing pro-inflammatory cytokines and existing as ectopic adipocytes in the cells of the alveoli.
Be sure to read the Disclaimer!
#2020-McGregor and #2021-Chauss
COVID-19 patients need vitamin D to ensure Th1 lymphocytes in their lungs shut down and so reduce inflammation
You may like to read my illustrated explanation of vitamin D autocrine
and paracrine signaling, https://VitaminDStopsCOVID.info/02-autocrine/
which is how almost all of vitamin D's functions are carried out.
This is unrelated to hormones - vitamin D has only one hormonal
function: to regulate calcium-bone metabolism.
I regard this as the most important COVID-19 article of all.
Autocrine vitamin D signaling switches off pro-inflammatory programs of Th1 cells
Daniel Chauss, Tilo
Freiwald, Reuben McGregor, Bingyu Yan, Luopin Wang, Estefania
Nova-Lamperti, Dhaneshwar Kumar, Zonghao Zhang, Heather Teague, Erin E.
West, Kevin M. Vannella1, Marcos J. Ramos-Benitez, Jack Bibby, Audrey
Kelly1, Amna Malik1, Alexandra F. Freeman, Daniella M. Schwartz, Didier
Portilla1, Daniel S. Chertow, Susan John, Paul Lavender, Claudia
Kemper, Giovanna Lombardi, Nehal N. Mehta, Nichola Cooper1, Michail S.
Lionakis, Arian Laurence, Majid Kazemian and Behdad Afzali
Nature Immunology 2021-11-11
This is the peer-review journal result of a preprint from July 2020:
As befits an article of this significance, there is a Research Highlight in Nature Reviews Immunology:
Here are some comments on this Research Highlight
I am being pernickety since so much (general health and suppressing
COVID-19 pandemic transmission, harm and death) depends on a clear understanding of vitamin D based autocrine signaling
. Chauss et al.
is a tremendous addition to our understanding, but the whole vitamin D
field is bedeviled by confused and confusing terminology, and to some
degree by the mistaken belief that immune cells need more hormonal
(blood-borne) 1,25-hydroxyvitamin D, when what they need is good
supplies of 25-hydroxyvitamin D. (https://vitamindstopscovid.info/05-mds/#calcifediol-not-calcitriol
- As is common in the literature, the term "vitamin D" is used
collectively to refer to three compounds: (vitamin D3 cholecalciferol,
25-hydroxyvitamin D calcifediol and 1,25-dihydroxyvitamin D calcidiol).
This is fine. However, the article also uses "vitamin D" in some
contexts where the specific compound should be identified.
- Many people won't understand clearly that "the final activation step of vitamin D" means "hydroxylating 25-hydroxyvitamin D to become 1,25-dihydroxyvitamin D" or that "inactive vitamin D"
means "25-hydroxyvitamin D". The terminology is a minefield,
since the "vitamin D receptor" only responds strongly, and in most
cases in any significant way, to 1,25-hydroxyvitamin D, rather than to
the other two compounds. Arguably it should be called the
"calcitriol receptor", but that would be confusing too, since it is an
essential molecule in the various chains of (general, collective)
"vitamin D" signaling (hormonal, autocrine and paracrine).
This problem of not specifically identifying the compounds was identified by Reinhold Veith in 2004:
- I think many people would not clearly understand: "Vitamin D also induced genome-wide changes in histone acetylation and recruitment of transcription factors, including at STAT3, IL10 and BACH2 loci". A more specific description - which would not fit within the article's presumably one page space allocation - would be:
Once the vitamin D based autocrine
signaling system is activated by a sufficient level of extracellular
complement factor C3b causing the 1-hydroxylase enzyme and VDR to be
produced in the cytosol, this enzyme hydroxylates 25-hydroxyvitamin D
(which must be present in the cytosol in sufficient quantities) to
1,25-dihydroxyvitamin D, each molecule of which promptly binds to a VDR
molecule. These bound complexes enter the nucleus where they bind
to retinoid X receptors and the triple complexes initiate a complex
series of histone, DNA and vitamin D response element interactions
which alter how a large number of genes are transcribed to mRNA.
In general principle these mechanisms are the same in all kinds of cell
which utilise vitamin D autocrine signaling as part of how so each cell
responds to its changing circumstances. The particular details of
what initiates the autocrine signaling system, and the specific details
of which genes have their transcription up- and down-regulated vary
from one cell type to the next. In the case of these Th1
lymphocytes, the researchers identified that successful completion of
the autocrine signaling cascade resulted in the upregulation of 262
genes and the downregulation of 128. Among the cellular
changes which result: reduced production of pro-inflammatory IFNγ;
increased production of anti-inflammatory IL-10 and IL6 and
increased production of transcription factors including JUN, BACH2 and
- ". . . clinical trials
may help shed light on whether there is clinical benefit to using
vitamin D as an adjunct therapy for COVID-19". Indeed they do: https://vitamindstopscovid.info/05-mds/ and https://vdmeta.com
. Chauss et al. provides a detailed mechanistic explanation for
why boosting 25-hydroxyvitamin D levels enables the immune system to
properly regulate otherwise destructive levels of inflammation.
Similar improvements to autocrine and paracrine signaling in other
types of immune cell are presumably the main reason why innate and
adaptive responses to pathogens also depend so strongly on good
25-hydroxyvitamin D levels: https://vitamindstopscovid.info/02-autocrine/#04-quraishi .
- Finally, it is a challenge to craft a heading to describe this
work. If space were not so scarce, a better title might be "Th1
lymphocytes need good levels of 25-hydroxyvitamin D in order that their
vitamin D based autocrine signaling system can work properly, enabling
each cell to respond to high levels of complement factor C3b and so
transition from its pro-inflammatory startup program to its
anti-inflammatory shutdown program".
The following section is based on
McGregor et al. preprint
. I am not sure when I will have time to fully read
and understand Chauss et al. My initial impression is that it
elaborates on but does not contradict what was in McGregor et al.
So please use the following summary of McGregor et al. as a rough guide
to Chauss et al. subject to the following caveats:
- This summary is of a preprint, while the full article is more
elaborate, refined and considered - with further experimental
- This summary was written by an electronic technician and computer
programmer who knows only a tiny fraction of the immunology and cell
biology which would be required to fully understand and appraise these
The following box repeats my summary from the list of recent research ../#lr
and in the 02-autocrine page. Below the box is a more detailed
summary of the cellular mechanisms described in this article.
Th1 lymphocytes isolated from the lungs of patients with
severe COVID-19 symptoms have an autocrine (https://vitamindstopscovid.info/02-autocrine/) signaling pathway
which should be
activated by high levels of complement (WP), to turn these cells off their initial hyper-inflammatory
program which produces pro-inflammatory IFNγ (interferon_gamma WP
which has antiviral and anti-bacterial activity as well as stimulating
inflammation: cell destruction such as by natural killer cells WP) and instead cause them to produce the anti-inflammatory cytokine IL-10.
(The cells always produce both these cytokines, but this transition to
a shutdown, anti-inflammatory program, involves less IFNγ and a lot
this anti-inflammatory pathway is not working in the Th1 cells from patients with severe COVID-19, due solely to insufficient 25hydroxyvitaminD3 = 25OHD = calcifediol for each cell's autocrine signaling system to function. (Until 2021-03-01 I mistakenly stated that the Th1 cells initially produced IL-17 - and that the experimenters restored the Th1's anti-inflammatory pathway by adding 25OHD in-vitro.)
This is a molecular and cellular explanation for why people with
low vitamin D have wildly dysregulated, overly-inflammatory (cell
killing), self-destructive immune responses. Such responses drive
sepsis, severe influenza, Kawasaki disease (KD WP), Multisystem Inflammatory
Syndrome (MIS discussion) and of course severe COVID-19. (See Paul Marik's explanation https://www.evms.edu/covid-19/covid_care_for_clinicians/ of how it is the immune response, not the virus, which causes the escalation to severe symptoms and death. See ../#2015-Stagi for research which shows KD children have very low 25OHD vitamin D levels.)
In severe COVID-19, severe inflammation in the lungs damages
endothelial cells (the inner lining of blood vessels and capillaries WP)
leading to hypercoagulative blood, causing microembolisms and larger
clots all over the body, which cause most of hypoxia, lasting harm and
It is not known whether the cause of all
the hyper-inflammatory immune system dysregulation - which causes some
COVID-19 sufferers people to develop
severe symptoms - is primarily the failure of these Th1 lymphocytes to
switch from being pro- to anti-inflammatory,
or whether this endothelial cell destruction etc. is also driven to a
significant degree by similar failures in the autocrine signaling
systems of many other
types of regulatory and/or directly anti-pathogen immune cell.
However, the determination of
the exact mechanism of failure in Th1 cells, in the context of such failures likely
also occurring in other cell types, is an extraordinarily valuable
contribution which deserves to be very widely known.
vitamin D levels (low circulating 25OHD, produced in the liver
from UV-B-generated and/or ingested vitamin D3 cholecalciferol) are
well known to reduce the effectiveness of numerous direct,
anti-pathogen, responses by the innate immune system cells and to
hinder the creation of antibodies for adaptive immune responses.
immune functions of vitamin D 25OHD are due to it being needed, in the
circulation, at higher levels than are sufficient for bone health
(sufficient for the kidneys to produce their much lower concentration
of circulating - and so hormonal - 25OHD), to
supply the autocrine / paracrine (inside the cell / to nearby cells)
signaling systems of all types of immune cells. All types of
cell can express the vitamin D receptor - and this is for
autocrine/paracrine signaling - not for responding to the much lower
levels of circulating 1,25OHD which regulates
calcium-bone metabolism. https://vitamindstopscovid.info/02-autocrine/#02-nothorm .
See http://aminotheory.com/cv19/#2020-Fabbri [B] for why 40ng/ml or more 25OHD is required for these autocrine signaling systems to function properly. See also the Quraishi et al. graph https://vitamindstopscovid.info/02-autocrine/#04-quraishi
which suggests that innate immune cell responses which fight bacterial and perhaps fungal infections keep improving,
presumably due to faster and stronger autocrine/paracrine signaling, as
25OHD levels rise, up to about 55ng/ml.
Please also see ../#25plusD3
for my suggestion of oral calcifediol (25OHD) plus D3 as the best
treatment for hospitalised COVID-19 patients, since this raises
circulating 25OHD to the levels needed for autocrine / paracrine
signaling in a few hours, rather than in the several days to a week
with vitamin D3.
For a more detailed summary of the McGregor et al. article, please see https://aminotheory.com/cv19/icu/#2020-McGregor .
Very strong clinical evidence of the importance of rapidly raising
circulating 25OHD levels hospitalised COVID-19 patients can be found the Cordoba calcifediol (25OHD) RCT: Castillo et al. 2020: ../#2020-Castillo .
Here is a more detailed summary of the cellular mechanisms
reported in this article - but please remember that this is the best effort of an electronic technician and computer programmer,
trying to summarize a dense cell biology preprint
reporting on an extensive project conducted by 25 researchers. What I have written below goes beyond a summary
and includes elements of critique
(regarding terminology) and some commentary
- so please parse it carefully and refer to the article itself, rather
than to what I write here, for your final decisions on the veracity and meaning of this research.
The wide positioning of the A
on the above graph exaggerates the relative changes in the degree
to which these two cytokines are created. (The horizontal and vertical
dimensions of Fig 1f are for the rates at which the genes for these two
cytokines are transcribed, which is approximately proportional to the
rates at which the cytokines themselves are produced.)
I wanted to know the relative concentrations of the two cytokines
produced by the cells between the B
activated, inflammatory, state and the D
shutdown, anti-inflammatory state. This can be estimated from each of two sets of data.
Firstly, the graphs in Fig 2C show the levels of the two cytokines
produced when there is zero 1,25OHD supplied to the cells, and then the
levels when three different concentrations of 1,25OHD are
supplied. I judge the 10nM supply to be sufficient to fully
activate the VDRs as they would be by normal, full, activation of the
autocrine signaling system with sufficient 25OHD supplied. So,
for instance, for the top IFNγ curve of Fig 2c, I can divide the ~5900
pg/ml shutdown D
level (4th red dot from the left) by the ~9,800 pg/ml B
initial, inflammatory, level and get a D
shutdown/inflammatory ratio of 0.6.
Secondly I can do the same for Fig S5d (in the separate Supplementary
Materials PDF) which represents the same things, but this time in
response to the experimenters supplying 25OHD so the real, already
activated, autocrine signaling system can operate, producing its own
1,25OHD to activate the cell's VDRs. Here, I chose the 3rd dot
from the right, because I assumed, perhaps wrongly, that the 50nM
(20ng/ml) 25OHD concentration was sufficient for the autocrine
signaling system to operate fully. For IFNγ in this graph I
estimate 7100 / 9100 (why not 9800 as before adding 1,25OHD?) = 0.78.
I estimate that
shutdown/inflammatory ratios are, from the two techniques respectively (first added 1,25OHD and second added 25OHD) to be IFNγ
0.6 & 0.78, and IL-10
2.5 & 1.4 (ignoring the vertical scale discrepancy, which I assume
is a typo). Neither of these experimental techniques tell us
exactly what quantities of the two cytokines Th1 cells produce in-vivo,
but it is the best we can do.
The normal, healthy, behaviour of Th1 lymphocytes to SARS-CoV-2 infection is as follows
Those isolated from the lungs of severe COVID-19 sufferers did not
transition to the anti-inflammatory program, and kept producing the
pro-inflammatory IFNγ, presumably due to lack of sufficient 25OHD in
those patient's bloodstream and therefore interstitial fluid, or
whatever fluids the cells were bathed in.
This discussion concerns T helper lymphocytes (WP
which developed from Th0 program cells into Th1 types, were attracted
to a site of infection - in this case the lungs of hospitalised
COVID-19 patients - and there multiplied (AKA, I think, "differentiated") so they
are present in much higher than numbers at this site than when they first arrived.
These Th1 cells have been activated (described below, perhaps before
they multiplied in number) and so are in their pro-inflammatory B
state (Fig 1f in the article and in my infographic above), producing their higher level of IFNγ (WP
) and their lower level of anti-inflammatory IL-10 (WP
We are interested in understanding how they transition from this state, firstly to a temporary state C
in Fig 1f, in which they produce high levels of both IFNγ and IL-10, and then to their final, anti-inflammatory, state D
of low IFNγ production and high anti-inflammatory IL-10. This is
the anti-inflammatory "shutdown program" referred to in the
title of the article.
Messenger RNA analysis (scRNA-seq WP
of Th1 cells from the lungs of hospitalised COVID-19 patients and
healthy controls revealed in the patient's Th1 cells, elevated levels
of mRNAs associated with the production of both IFNγ and complement.
Compared to the mRNA expression patterns in the T cells found in BALF
(bronchoalveolar lavage fluid) of controls, patients' patterns were
skewed to Th1 as opposed to the Th2 and Th7 lineages. However, no
such skew was observed in T cells from peripheral blood:
that expression of the Th1 program is a specific feature of Th cells at
the site of pulmonary inflammation where virus-specific T cells may be
High levels of complement production in Th1 cells has been observed in
lung infections and specifically with SARS-CoV-2, which is known to
particularly induce the production of complement factor C (C3). A
fragment of this, C3b, binds to the CD46 receptors (WP
found on the plasma membranes of human (but not mouse) T cells.
When all is working well, with sufficient 25OHD, drives their shutdown from the initial pro-inflammatory B
state to the final anti-inflammatory D
The switch from effector
[such as these Th1 cells in state B
which produce complement and IFNγ, which is an antiviral compound which
also stimulates ideally healthy cell destruction AKA inflammation] T
cells, important for pathogen clearance, into IL-10 producing cells
reduces collateral damage and is a natural transition in a T cell’s
life-cycle. This suggests that IL-10 is produced by cells that are
successfully transitioning into the Th1 shut program. Indeed, in
models of T. gondii [WP] and T. cruzi [Chagas disease WP] infections, mice unable to produce IL-10 clear infection more rapidly but die of severe tissue damage from uncontrolled Th1 responses.
IL-10 mRNA in patients' BALF was found at about 1/4 the level it was
found in controls, consistent with the hypothesis that Th1 cells in the
lungs of hospitalised COVID-19 patients indeed did not initiate - or at least complete - their
Activation of CD46 receptors by C3b upregulated 24 transcription
factors (TFs). (Genes for these 24 were found to have been
transcribed to mRNAs at higher levels than without this CD46
activation.) One of these TFs was for the vitamin D receptor
(VDR) and another was the CYP27B1 gene for the 1-hydroxylase enzyme,
which converts intracellular 25OHD AKA calcifediol (diffused there from
circulating 25OHD) into intracellular 1,25OHD AKA calcitriol which
binds to and activates the VDR.
So we see that this CD46 activation initiates the first steps of vitamin D autocrine signaling https://vitamindstopscovid.info/02-autocrine/
, while the other transcription factors drive other cellular responses
in parallel to (if all works well) the effects of this autocrine
signaling process, which might also involve this locally produced
1,25OHD diffusing to nearby cells as a paracrine agent.
Although both vitamin D based autocrine and paracrine signaling had
been previously described in Th1 cells, this research project set out
to elucidate the molecular mechanisms and functional consequences of
this, which were previously unknown. They did a great job!
(Page 5 starts with some
descriptions of CD3 CD28 activation driving internal production of C3b
which binds to CD46 on the cell surface, which is an autocrine
signaling process, quite separate from the vitamin D autocrine
signaling process we are most interested in. I don't understand
this enough to summarize it.)
Sidebar on vitamin D terminology and "vitamin D is a hormone":
As is common in many vitamin D articles, the article states that "vitamin D is a steroid hormone". Here we get into terminological and factual difficulties
, which I discuss at: https://vitamindstopscovid.info/02-autocrine/#02-nothorm
In some articles, "vitamin D" means specifically vitamin D3
cholecalciferol, with 25OHD and 1,25OHD being classed as "vitamin D
metabolites". However, this makes no sense since the receptor
universally known as the "vitamin D receptor", for all (or at least
most) practical purposes, is only activated by 1,25OHD.
Some other articles use "vitamin D" to refer collectively to the three
compounds: D3 cholecalciferol, 25OHD calcifediol and 1,25OHD
calcitriol. This makes sense to me, and this is how I use the
term. Some articles use "vitamin D" to mean both these incompatible definitions, without
any indication that this is invalid - which is very confusing.
This article classes the artificial compound 1,OHD alfacalcidiol [WP
which can be converted to 1,25OHD by the 25-hydroxylase enzyme normally
found in the liver (which normally converts D3 to 25OHD) as both an
of vitamin D, which I am pretty sure it is not, and simply as
D, which it is not. Alphacalcidol is an analogue of
"vitamin D", where this is the collective use of the term, since it is
similar to D3 (an extra hydroxyl group at position 1) and can be
converted to 1,25OHD by adding another hydroxyl group at position
25. As far as I know, it is an artificial substance not found
Generally the article (and this is a preprint) uses the term "VitD" to
refer to one of the three compounds, usually 1,25OHD, but sometimes
25OHD, which I think is confusing. It would be better to refer to
the specific compounds, in all cases.
1,25OHD, when in circulation in the blood, acts as a hormone: endocrine
cell-to-cell signaling between cells anywhere, or in many distant
places in the body, with the compound being circulating in many parts
of the body via the bloodstream.
This does not mean that 1,25OHD is
a hormone. It can act as a hormone when in circulation. Nor
does it make any sense to state that "vitamin D" is a hormone, with
this meaning either just D3 or collectively D3, 25OHD and
However, this "vitamin D is a hormone" statement and this confused and
confusing use of terminology is industry standard practice, since
it is so common in vitamin D research articles, including a recent
articles co-written by the acknowledge leader of the field, Michael Holick:
Immunologic Effects of Vitamin D on Human Health and Disease
Nipith Charoenngam, Michael F. Holick 2020-07-15
Nutrients 2020, 12(7), 2097
which I think is a great article, apart from these terminological problems.
(Page 5 to 6.) The researchers prepared activated*
Th1 cells and treated them with 1,25OHD to determine which genes were
upregulated (296) and downregulated (157) by this robust VDR
activation. (The precise details of these transcriptional - DNA to
mRNA - changes would depend on the exact state of the cells, since
there all sorts of subtleties in the direct mechanisms of
transcription, and, for instance, acetylation of histones in ways which
also affect the rate at which particular genes are transcribed.) Among
these transcriptional changes were:
- Less transcription ("repressed") of the IFNG (interferon gamma) gene which, when
copied to mRNA, causes the protein making machinery (ribosomes) to
- Likewise, repression of the gene for the pro-inflammatory cytokine IL-17 [WP].
- More transcription ("induced") of the genes for IL-10, IL-6 (generally, but not always, a pro-inflammatory cytokine [WP]) and several transcription factor genes including JUN (for c-Jun [WP]), BACH2 (for the "broad complex-tramtrack-bric a brac and Cap'n'collar homology 2" protein [WP]) and STAT3 (Signal transducer and activator of transcription 3 [WP]). I discuss these further below. (It is 1AM and I can feel myself, very slowly, becoming a cell biologist - though I would not want to be examined on that BACH2 thang.)
The induction of IL-6 was a surprise to the researchers, since this is
a frequently encountered and typically pro-inflammatory cytokine.
However, as I best understand it, it seems that in these circumstances
IL-6 is being produced for purposes of internal signaling (autocrine, I
guess) rather than to stimulate high levels of inflammation in the
vicinity of this cell.
this context means that the T cells had C3b bound to their CD46
receptors which initiated the transcription of the genes for VDR
(vitamin D receptor) and the CYP27B1 1-hydroxylase enzyme, both which
were made as a result. I assume that the medium in which
these cells were tested had little or no 25OHD, since this would have
been converted to 1,25OHD, which would have bound to the VDR and so
caused the repressed and induced patterns of mRNAs described above and
The researchers repeated these tests and found the same patterns of
gene transcription when, instead of directly adding 1,25OHD, they added
25OHD (calcifediol). This shows that the activated cells, when
they had enough 25OHD for the activation-created CYP27B1 1-hydroxylase
enzyme molecules in their cytosol to convert to 1,25OHD, which then
binds to the VDR molecules there (also created by the
the bound complex of 1,25OHD and VDR "migrates" (or does it simply
diffuse?) to the nucleus, complexes with some other molecules and the
resulting ensemble changes gene expression as
expected so the cells transition successfully from their initial
pro-inflammatory state B
, through their pro- and anti-inflammatory state C
, to their shutdown mode: anti-inflammatory state D
We see from this that if all the cell's internal mechanisms are functioning normally, that successful transition from B
involves these external
factors and these internal
- High levels of complement
- perhaps created in part by the population of Th1 cells in the same
vicinity (millimetres, I guess) which are in the same initial B
state, (or is some or all of this complement, C3 protein at lest, made inside the Th1 cell??) lead to (by some
processes I am not trying to include in this summary) to the C3b part
of these complement compounds binding to the cell's CD46 receptors,
with their binding site on the outside of the cell's plasma
- The activated CD46 receptor
alters (by means I am not trying to summarize) gene expression in many
ways, including by inducing (increasing the number of mRNA copies of)
the genes for the CYP27B1 1-hydroxylase enzyme and VDR.
These two of the many other gene expression altering changes caused by the activation of the CD46 receptors initiates this cell's vitamin D based autocrine (and potentially paracrine) signaling process.
This process is initiated in a response to this particular cell's
circumstances - it is not some kind of homeostasis-maintaining feedback
loop. Each cell type has similar
principles to steps 2 to 4, but the details of the initiating
process and of which genes are induced and repressed are completely
different between the cell types.
I think of this as the initiation
step of this cell's vitamin D based autocrine signaling system.
(I am leaving out various mRNA editing steps, if such steps occur with
these genes - such as splicing removing introns - see
post-transcriptional modification [WP].)
- These mRNAs leave the nucleus, get into the cytoplasm and there
direct ribosomes to make both the CYP27B1 1-hydroxylase enzyme and VDR
proteins. This is translation [WP],
but just of these two proteins (always the same for every cell which
uses vitamin D based autocrine / paracrine signaling).
(Meanwhile, other gene transcription changes caused directly by CD46
activation also result in more or less of other proteins being made,
but this is not part of the vitamin D autocrine signaling process we
are focusing on at present.)
- Externally supplied 25OHD (25 hydroxyvitamin D3 AKA calcifediol) is necessary to the next step. The cell cannot make its own 25OHD. Even if D3 is present in the interstitial fluid [WP]
(or perhaps the plasma, if the cell is in the bloodstream) it lacks the
25-hydroxylase enzyme to convert it to 25OHD. Generally we
assume that UV-B produced or ingested D3 is converted, over days
to a week or so, in the liver by this enzyme into 25OHD, where it goes
into circulation in the blood plasma (more on binding proteins
best I understand it, circulating 25OHD diffuses from the plasma
into the interstitial fluid, without any particular active transporters
or energy expenditure. From there, it diffuses - again without
transmembrane transporters, energy expenditure or any other directional
processes - from the cell's plasma membrane and into its
cytosol. As far as I know, the 25OHD molecules largely, but not
entirely hydrophobic - only two hydroxyl groups and all the other
outside parts of the molecule being oliophilic [WP] hydrogen atoms and small. I assume the molecule makes its own way across the plasma membrane. Can anyone
provide more details or confirm this?
Inside the cell, 25OHD is subject to degradation by the 24-hydroxylase
membrane, which is encoded by the CYP24A1 gene, the name of which is
often applied to the enzyme itself. [WP].
(This is all basic vitamin D stuff - not specific to cells with vitamin
D based autocrine / paracrine signaling systems.)
The 24-hydroxylase enzyme is inside the mitochondria [WP]
and its molecular numbers, or at least its overall activity in the
body, is upregulated by high 25OHD levels. (The diagram https://www.wikipathways.org/index.php/Pathway:WP1531
shows this enzyme degrading 25OHD, here referred to as calcidiol, and
being upregulated only by parathyroid hormone and 1,25OHD calcitriol,
whether circulating or perhaps locally produced, rather than
25OHD. However, my potentially faulty understanding is that its
activity is also driven by 25OHD itself, in a self-regulatory system
which causes the curved D3 or 25OHD input to 25OHD level response in
the Ekwaru et al. 2014 diagram: https://vitamindstopscovid.info/01-supp/a-ratios/ .)
This converts some
25OHD to 24-25OHD which is an irreversible operation, leading to the
latter being metabolised and its components excreted. This is the
primary, or perhaps sole, self-regulatory mechanism which tends to
limit 25OHD levels if there is a large input of D3 (and/or its UV-B
creation) or (artificially) 25OHD into the body.
I mention this 24-hydroxylase enzyme firstly because this intracellular
degradation of 25OHD coming into the cell, by diffusion, is both part
of a body-wide limiting system but also a means for limiting high 25OHD
levels in this individual cell.
Secondly, this enzyme also degrades 1,25OHD, which has a much shorter half-life than 25OHD. (I
guess this shorter half-life is due primarily to the greater affinity
of 1,25OHD for this 24-hydroxylase enzyme, but this is the limit of my knowledge and I
don't have time now to dive into another rabbit hole to find out for
This externally supplied 25OHD (or that which remains
subject to 24-hydroxylase activity AND it being already consumed as
described next) finds its way to the active site of the 1 hydroxylase
enzyme. The concentration of 25OHD in the cytosol is very low -
so there is probably only one such molecule every 320 nanometres cubed
(my rough calculations at https://vitamindstopscovid.info/02-autocrine/#03-minlev from which the next illustration is drawn) when the molecule, in red below, is only 0.2 nanometres long.
The 25OHD molecule also relies on thermal vibrations and some local
electrostatic attraction to rotate it into the correct orientation and so
slot into the active site correctly. I mention this to highlight
that this reaction proceeds at a rate limited by the concentration of
25OHD, which is very low, and the likely still lower concentration of
the 1-hydroxylase enzyme. (Cofactors are necessary for the 1-hydroxylation
reaction, some of which are consumed in the process.)
The newly created 1,25OHD molecule leaves the active site and the
enzyme is ready for another 25OHD molecule to float into position.
The rate at which 1,25OHD production proceeds is directly dependent on
the 25OHD level in the cytosol, and this is reduced for every 25OHD
molecule converted, or lost to 24-hydroxylase.
For the autocrine signaling system to operate rapidly and fully, there
needs to be a substantial rate of 25OHD to 1,25OHD conversion, because
the latter is short-lived. This means there needs to be a
continual supply of 25OHD to the cell, by diffusion from the
interstitial fluid (or perhaps directly from the plasma).
So the presence of adequate, externally supplied, 25OHD in the cytosol(which
should be the case at all times - this presence not created by the CD46
initiation of this autocrine / paracrine signaling
system) together with the newly created (by the initiation of the autocrine / paracrine signaling process) 1-hydroxylase enzyme leads directly to the outcome of this step: in-cytosol production of 1,25OHD.
Some of this 1,25OHD is degraded by 24-hydroxylase - over time, most of
which remains in the cell is degraded in this way. Some of this
1,25OHD diffuses from the cell and may act on nearby cells as a
Some of this 1,25OHD drives the next step.
- The newly created 1,25OHD molecules in the cytosol soon find themselves bound to the VDR, for which they have a very high affinity. There are a bunch of details regarding how the complex of the two "migrates"*
to the nucleus, binds with other things and (by various mechanisms I am
far from understanding - and I am not sure if anyone fully understands
them all) the presence of large numbers of bound VDR complexes alters
the transcription (AKA expression) of numerous genes. The exact
details of which genes are induced or suppressed, and to what degree,
depends entirely on the cell type.
"Migration" is the usual term, but as far as I know there is no active,
powered, directional mechanism for this. Can anyone improve my
knowledge? I wrote "migrate" to be compatible with all I have
read, but for now I assume the bound complex diffuses around the
place randomly, with some of them getting into the nucleus.
This is the transcription phase of the autocrine signaling system.
There is some kind of degradation process for bound VDR complexes in
the nucleus, so they don't accumulate and alter gene translation
forever. I don't know the details, but I guess the VDR itself is
retained and is free to diffuse back into the cytosol. However,
there is surely a degradation process for the VDR as well, otherwise
they would accumulate and this part of the autocrine signaling system
would not be turned off when the initiating condition for the system is
no longer active. The same goes for the 1-hydroxylase enzyme.
- The changed mRNA numbers for the various genes, by altering the protein produced by ribosomes, alters the function of the cell.
This is the final output of this entire vitamin D driven autocrine
signaling process for this particular type of cell.
mRNAs are rapidly degraded, and proteins (which may be exported or
transformed in various ways) don't last forever. So for the
autocrine signaling system to continue to function in its activated
state, the original initiating stimulus (in the Th1 cell type, C3b
binding to its CD46 receptors) must
continue to be present, 25OHD needs to be continually supplied, by
diffusion, so 1.25OHD production can continue. So there needs to
be a continuing supply of 25OHD, by diffusion from the bloodstream into
the cytosol of each cell.
The exact details by which IFNγ and IL-10 are produced by these Th1
lymphocytes is somewhat more complex than getting their mRNAs into the
cytosol. More details of this were discovered by the researchers
and are summarized below. However, these details do not alter the
central role of vitamin D based autocrine signaling controlling the
Th1 cells' production of these to crucially important cytokines other
than that they also rely to some extent on the vitamin D based
autocrine signaling system also upregulating the gene which produces
This has been a rather detailed excursion into the molecular details of
the central (common to all cell types which use it) mechanisms of
We need to think about this because at present (March 2021) the world
is going to hell in a handbasket, in large part due to most humans
having insufficient 25OHD in the cytoplasm of their immune cells to
initially fight the SARS-CoV-2 infection, and in particular, in the
context of humans' genetic predisposition to overly inflammatory immune
responses due to lack of helminths:
If everyone had about 50ng/ml
or more (twice is just fine) 25OHD in
their bloodstream, all these immune cell types, Th1s included, and all
other cells in the body which use vitamin D autocrine (and perhaps
paracrine) signaling, would be working pretty well. Omega-3 and
other nutrients such as boron (#08-boron
) and vitamin C are also important, but for
now we focus on 25OHD, since this is the most important nutritional
deficiency which clobbers all aspects of the immune system.
The present article concerns low 25OHD greatly increasing the (already
genetically driven) overly-inflammatory pattern of immune responses -
and this response (perhaps just from these Th1 cells, but likely
involving weak and dysregulated responses from all types of immune
cell) is the primary driving reason why some COVID-19 sufferers
progress to severe symptoms, with high levels of endothelial damage,
which causes the hypercoagulative blood which does the real damage with
microembolisms and larger clots all through the body.
Inquiring minds now want to know how the (ideal, as far as we know ~50ng/ml 125nmol/L
or more 25OHD blood levels relate to the levels of 25OHD in the cytoplasm of
the Th1 cells (and all other cell types which also use vitamin D for
autocrine / paracrine signaling). See my pages at the current
site and at https://vitamindstopscovid.info
for research which shows we need such levels, at least for autocrine
signaling to work properly. (Some people, with auto-immune diseases,
need two or three times these levels to significantly reduce or abolish
their symptoms - here I am discussing most people, not these. See
the McCullough et al. article mentioned above for examples.)
Looking at the experimenter's graphs for the IFNγ and IL-10 responses
to 1,25OHD (Fig 2C) and 25OHD (Fig S5d in the Supplementary data PDF),
we see that most of the change occurs between concentrations of 0 and:
1,25OHD: 1nM, with about half the action at 0.1nM. (However, I chose 10nM for the levels which I think reflect the complete transition to anti-inflammatory state D.)
25OHD: (My guess, based on interpolating between the 0, 10 and 50nM levels.) 25nM with about half the action around 7nM.
nM == nano Mols == nmol/L = a billionth of a mole of molecules per litre.
1 nanogram per millilitre of D3, 25OHD or 1,25OHD is the same concentration as 2.5nmol/L of these compounds.
A Mole [WP
] is 6.022 x 1023
molecules. 1 nanomol is 6 x 1014
molecules, so 1nM = 1nmol/L = 0.4ng/L means that for each molecule, there is 1.66 x 10-15
litres of water. This volume is a cube with sides 1.185
micrometres = 1185 nanometres, which is big for a molecule which is
only 0.2 nanometres long. This is something like one 1mm
long object in a home swimming pool.
The nM concentrations reported in these graphs are for the solution in
which the researchers bathed the cells, and for now we assume reasonably
free diffusion into the much smaller cell bodies - so these figures
roughly represent the concentrations in the cytosol of these Th1
So it seems that to get the autocrine signaling system of these cells
to respond fully, we need about 25nmol/L 25OHD in the cytosol, which is
I think the discrepancy between this very approximate figure and the abovementioned ~50ng/mL
for circulating 25OHD in the bloodstream can be accounted for roughly as follows:
- Most of the 25OHD in the blood plasma is bound tightly to vitamin D binding protein (DBP) molecules [WP],
which with 458 amino acids, dwarf the 25OHD molecule which is not much
bigger than a single amino acid. There is is considerable
individual and racial variation in the form of these molecules and in
the concentration in which they are present in the plasma.
Although it is possible to measure the level of 25OHD which is not
bound to DBP, this is not a commonly available from pathology labs,
although such measurements would probably be more clinically relevant.
DBP also binds D3 and the low level of circulating (hormonal) 1,25OHD.
- Most of the 25OHD which is not bound to DBP molecules is bound - much less strongly, I recall - to albumin proteins [WP].
So the fraction of 25OHD available to diffuse into the interstitial
fluid, and then into the cytosol of immune cells such as these Th1
lymphocytes, is the unbound fraction plus some part of the albumin-bound
- At any intracellular parts of the 25OHD molecules paths of diffusion from
the plasma to the cytosol of the Th1 lymphocytes (such as diffusing
through endothelial and other cells of capillaries), there will be some
losses due to the actions of the 24-hydroxylase enzyme.
In March 2021 we know, from numerous research articles in the last
year - many of which are analysed by some apparently expert, but
anonymous, souls at: https://vdmeta.com
, some of which are listed at https://aminotheory.com/cv19/#lr
- that people who succumb to severe COVID-19 have generally lower, on
average, 25OHD levels than those who don't. This becomes
particularly clear when we consider the dramatic improvements due to
giving hospitalised COVID-19 patients in Cordoba, Spain, just 0.532mg
of oral 25OHD calcifediol: https://aminotheory.com/cv19/#2020-Castillo
, raising their 25OHD levels from a likely 4
range to around 60ng/ml
in a few hours.
So lets think about D3 intakes and these low 25OHD levels for a
moment. For 70kg adults, 0.125mg 5000IU D3 a day maintains about
. This is 1 gram every 22 years
. These patients
lower 25OHD levels of 4
probably result from much lower total quantities of D3
from UV-B, food and few, if any, supplements. Let's say their D3
input was 0.01mg 400IU a day, with some patients, in recent years at
least, with even less, due to being aged, being indoors, having dark
skin, covering their bodies almost entirely when outside etc. The
UK government recommends 0.01mg 400IU D3 a day for adults - in winter
These people would generally not be patients in hospital with
COVID-19 if they had been supplementing 0.125mg D3 a day (or more
according to bodyweight, with more for people suffering from obesity: https://vitamindstopscovid.info/01-supp/
So this severe ill-health, and numerous other aspects of ill health, has
been in large part caused by these patients having disastrously low D3
intakes (though generally about enough to ensure they don't get rickets
or unusual levels of osteoporosis), of about 1 gram every 100,000 days
= 274 years
. Pharma grade D3 costs about USD$2.50 a gram ex
factory in 1kg quantities. (That said, UK autocrine signaling pioneer Martin Hewison states in a 2021-03-03 video
that the UK is the vitamin D deficiency centre of the world, and that
rickets is not uncommon in certain communities - presumably of people
with dark skin and/or who avoid direct sun exposure of their skin.)
This global epidemic of vitamin D deficiency has been running for
decades and centuries. Without it, there would be little or no
need for the disastrous lockdowns, social distancing, economic and
travel shutdowns etc. which governments have imposed to combat
COVID-19, and surely will continue to impose in the years as vaccine
manufacturers (and populations of entire countries) play cat-and-mouse,
or whack-a-mole, with increasingly efficient viral variants
are also under strong selection pressure to evolve avoidance of
existing infection- and vaccine-induced immune responses.
Back to the research article!
The researchers demonstrate further details of the Th1 immune shutdown
program, which I discuss below. This research shows that
the shutdown is dependent on adequate 25OHD levels in the cytosol of
the cell, since, in-vitro (as we surmise, in-vivo, from high 25OHD
levels apparently causing a reduction in this hyper-inflammatory immune
dysregulation) the cells do not respond
to high levels of complement by transitioning from their
pro-inflammatory program to their anti-inflammatory shutdown program, unless sufficient 25OHD is supplied to them
This concludes the section of my summary and discussion which is most
relevant to vitamin D autocrine signaling in Th1 lymphocytes.
What follows is an attempt to summarize some of the other molecular
details of exactly how IFNγ, IL-10 and IL-6 production is regulated in
these cells, within the context of these Th1 cells' vitamin D based autocrine
/ paracrine signaling system.
Again, please remember this is the best effort of an electronic
technician to understand and describe complex cell-biology
The researchers noticed that IL-10 production was proportional to IL-6
production. They added IL-6 to cells which had their CD46
receptors stimulated, and so the vitamin D autocrine signaling system
initiated, but with the cells stuck in their pro-inflammatory B
state due to there being no 25OHD supply. (With sufficient 25OHD
they would have converted some of it to 1,25OHD, completed the
autocrine signaling pathway and transitioned through C
to the anti-inflammatory D
state.) This caused the cells to produce pro-inflammatory IL-17,
which had been reported by other researchers. This seems
clinically relevant to me, since this is an additional mechanism for
pro-inflammatory dysregulation in patients with low 25OHD levels, IL-6
being at high levels in all these COVID-19 lung infections.
Both c-JUN and STAT3 transcription factors were produced by the full
operation of the vitamin D based autocrine signaling system (leading to
). c-JUN phosphorylation [WP
was also driven directly by this. STAT3 phosphorylation was
dependent not on this completion of the autocrine signaling
pathway, but on the presence of IL-6, the production of which was
directly caused by the completion of this pathway. They confirmed
that the IL-6 receptor was necessary for this process by introducing an
IL-6 receptor blocker Tocilizumab. (Imagine the dreams of the people whose job it is to come up with these names.)
Since this IL-6 was, at least part, produced inside the cell,
and activates receptors in the same cell, this is a separate IL-6 based
autocrine pathway with these IL-6 molecules acting as autocrine
agent. To the extent that some of the IL-6 which activates these
receptors was produced in nearby cells (Th1 or other types), those IL-6
molecules are acting as paracrine agents.
With further evidence from the cells of two patients with a rare STAT3 mutation (!) the researchers concluded:
mean completion of the autocrine signaling response to high levels of
complement, which is only possible with sufficient 25OHD] induces STAT3 and IL-6, and IL-6 phosphorylates STAT3 to promote production of [anti-inflammatory] IL-10.
Page 7 to 8 describes the researchers investigation of the molecular
details of how VDR activation (which will transition these cells from
state, through C
) work, such as by the VDR, complexed to other molecules, affecting the degree to which histones [WP
] are acetylated [WP
in particular places. The 46 chromosomes add up to 1.8 metres of
DNA which is wound around much smaller histone molecules. This
typically tight winding impedes access to most of the DNA by the
enzymes which can copy its information into messenger RNA molecules
(mRNAs). Acetylation in particular parts of the histone enables
looser DNA winding and therefore greater access of these enzymes to
particular parts of the DNA - including the genes which, in this
particular cell type, VDR activation increases the transcription
of. I don't clearly understand their observations regarding
c-Jun, BACH2 and STAT3 transcription factors, their phosphorylation and
super enhancer structures arising from histone acetylation.
BACH2 - a transcription factor whose production is increased "by
vitamin D" (meaning the successful autocrine process leading to state D
- is crucial to the increased transcription of other genes by this
process, including the gene for the IL-6 receptor which, as as noted
previously, is an essential link in the chain of IL-6 based autocrine /
paracrine signaling which leads to the production of anti-inflammatory
of VitD-repressed genes . . . was significantly higher in
bronchoalveolar fluid Th cells of patients with COVID-19 than
So the Th1 cells from COVID-19 patients did not have their (high levels
of complement driven) CD46 activated vitamin D based autocrine /
paracrine signaling function run to completion (from state B
presumably due to lack of 25OHD, leading to little or no production of
autocrine agent 1,25OHD, and so little or no activation of VDRs which,
if activated, would have repressed the transcription of these genes.
Here endeth the lesson. There will be a test on Friday!
Review article on Vitamin D and COVID-19
Vitamin D and COVID-19
Bilezikian, Daniel Bikle, Martin Hewison, Marise Lazaretti-Castro, Anna
Maria Formenti, Aakriti Gupta, Mahesh V. Madhavan, Nandini Nair1, Varta
Babalyan, Nicholas Hutchings, Nicola Napoli11, Domenico Accili, Neil
Binkley, Donald W. Landry, Andrea Giustina 2020-08-04
This is a review of observations and mechanisms. I haven't had a chance to read it yet.
Farid Jalali's hypothesis of vascular damage and shunting via diffuse pulmonary-bronchial anastomoses
Be sure to read this beautiful
detective work which offers the best explanation yet for many
perplexing aspects of COVID-19. The first link is to a video
podcast with PDF and discussion and the second is to a text and graphic
presentation with comments by Cameron Kyle-Sidell MD:
Cameron Kyle-Sidell's appreciative comments begin with:
I applaud Dr. Jalali for proposing a model for COVID-19 injury based on
seemingly sound physiologic principles. This model does provide
explanations for anecdotal observations made by bedside physicians
treating COVID-19 patients. For example, the presence of
dorsal-predominant shunting would explain why proning leads to marked
oxygenation improvement that is not sustained once the patient returns
to the supine position.
This work highlights the importance of protecting the integrity of the endothelium and limiting vasoconstriction
, which is caused by excessive angiotensin II, resulting from SARS-CoV-2 destroying the ACE2 receptors. See #2020-Kim
below regarding how important vitamin D 1,25OHD
is for protecting endothelial cells and inducing vasodilation
This is not necessarily just 1,25OHD in the plasma in general, but
could be released in a paracrine fashion (short distance signaling
between nearby cells [W
]) after it is produced internally (in part for autocrine [W
] signaling) and then diffuses out of the cell. Autocrine cell signaling (Khan Academy explanation
is a big part of how immune cells control gene expression, and with
leakage of the internally produced 1,24OHD, we have the possibility of
paracrine reception of this on membrane-bound vitamin D receptors of
nearby cells, such as the endothelial cells described by #2020-Kim
, where it would upregulate endothelial protection and nitric oxide mediated vasodilation.
What proportion of endothelial cell vitamin D receptors are on the
apical surface, and so exposed to plasma 1,25OHD, plus any locally
secreted by immune cells there? What proportion are on the other
side (outside the vessel or capillary = basolateral) and so, I guess
less affected by the normal bone and calcium regulating level of
1,25OHD as a hormone, and more affected by the actions of immune cells
in the surrounding tissue?
See also ../obesity/
According to the
research I review there, the excess adipocytes of obesity express ACE2
receptors and so can be infected with SARS-CoV-2. These are in
the outer lining of the heart and there are also ectopic adipocytes in the cells which make up the alveoli
I had never heard of these, but assuming it is true, then obese people
would have further infection in their lungs combined with extra
Farid Jalali's hypothesis of serotonin (5-HT) playing a major role in COVID-19 lung pathology
In late June 2020, Farid Jalali released a text-only document:
in which he proposes that serotonin, especially that released by
platelets in the clotting cascade, plays a major role in making things
much worse in SARS-CoV-2 infected lungs. He suggests that the
normal clearance mechanisms, which are in the lungs themselves, can no
longer function, so serotonin builds up in the whole circulation
causing further trouble.
As with the above diagrammatic hypothesis, I think only a handful of
people - pulmonolgists and the like - could fully appreciate and
critique what he is proposing.
However, I understand enough of it to envisage these as possible
explanations for the extreme levels of damage which occur with the
worst symptoms of COVID-19.
"Vitamin D" does not appear in either of these documents. The big
question is why is there so much inappropriate inflammation in these
patients. Surely low vitamin D levels - anything below 40ng/ml
250HD - plays a role in this immune system dysfunction. I also
suspect that low boron plays a role as well. See: ../#08-boron
Respiratory epithelial cells convert 25OHD to 1,25OHD
Further to the discussion above on
intracrine and paracrine signaling of 1,25OHD, here is a pertinent
article, from 2008. The lead author is a critical care
pulmonologist in Iowa.
. . . primary lung epithelial cells express high baseline levels of
activating 1α-hydroxylase and low levels of inactivating
24-hydroxylase. The result of this enzyme expression is that
airway epithelial cells constitutively convert inactive
25-dihydroxyvitamin D3 to the active 1,25-dihydroxyvitamin D3.
Also, a 2011 article:
Toll like receptors (TLRs) respond to extracellular fragments of
pathogens and/or of cellular damage, upregulate CYP27B1
(1a-hydroxylase) which converts 25OHD to 1,25OHD. This activates
the vitamin D receptor (the production of which is also upregulated by
the activation of TLRs) and the activated receptor travels to the
nucleus and upregulates genes specific to the particular cell
type. This diagram depicts some of the actions. (I have
another diagram somewhere depicting autocrine 1,25OHD signaling between
He suggests 25OHD levels of 40 to 60ng
, which I agree with.
Angiogenesis; ACE2-positive lymphocytes. alveolar and endothelial cells
Also of interest is this analysis of
COVID-19 damage to lung tissue differs from that resulting from
H1N1 influenza in 2009:
The lungs from patients with Covid-19 also showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with Covid-19 showed widespread thrombosis with microangiopathy. Alveolar capillary microthrombi were 9
times as prevalent in patients with Covid-19 as in patients with
influenza (P<0.001). In lungs from patients with Covid-19, the
amount of new vessel growth — predominantly through a mechanism of
intussusceptive [splitting] angiogenesis — was 2.7 times as high as that in the lungs from patients with influenza (P<0.001).
Are these new blood vessels or capillaries fed by the pulmonary or
bronchial artery? To what extent were they clogged with
microthrombi? To what extent could they participate in gas
exchange? Although the accompanying editorial
notes that the H1N1 samples are not directly comparable, this graph
indicates that angiogenesis is a very prominent feature in
COVID-19. This analysis is from autopsies - so this progression
is for the patients with the worst symptoms.
79 inflammation-related genes
were differentially regulated only in specimens from patients with
Covid-19, whereas 2 genes were differentially regulated only in
specimens from patients with influenza; a shared expression pattern was
found for 7 genes.
This suggests to me a high immune system involvement, in addition to
direct viral damage, in both the COVID-19 and H1N1 affected
lungs. Low vitamin D drives inappropriate, self-harming,
overly-inflammatory responses, as well as weak antiviral defense
Loss of ACE2 receptors is a prominent part of Farid Jalali's hypothesis
(above), driving excessive angiotensin II. To what extent is this
due to direct viral damage (virus attaches to ACE2 which is then
internalised with the virus and so no longer remains active on the cell
membrane) or due to immune system destruction of infected cells, and
perhaps those nearby even if uninfected?
Why, in these illnesses are ACE2 receptors more frequently found on
epithelial and endothelial cells? Are these cells sensing
excessive blood pressure due to microthrombi blocking the capillaries
and/or vasoconstriction there? If so, do they sprout ACE2
receptors in an attempt to reduce angiotensin II levels and so reduce
If so, then these extra ACE2 receptors make them vulnerable to viral
infection and the whole situation can spiral out of control.
|Fraction of cells with ACE2 receptors
Why do ACE2 receptors appear on lymphocytes? Are these lymphocytes also
acting to reduce local angiotensin II to reduce vasoconstriction?
If so, then the virus is well adapted: it gets into cells which already
express the ACE2 receptor, taking these receptors, and then the cells,
out of action. This reduces angiotensin II levels, driving
vasoconstriction and microembolisms which cause multiple cell types
which normally express few, if any, ACE2 receptors to express lots of
them, in an attempt to reduce vasoconstriction - so the virus can
infect them too. Inducing lymphocytes to express ACE2 and so be
infected is quite a twist.
This article seems be relevant, mentioning ACE2-positive lymphocytes,
but I have not attempted to pursue this line of inquiry further.
Please suggest pertinent articles and discussions. ../#contact
Vitamin D and Endothelial Function
Your COVID-19 patients are (according to Philippino and Indonesian research I cite here cv19/
generally seriously deficient in vitamin D. Surely you can
improve their chances of survival with vitamin D supplements.
Cholecalciferol (D3) takes days to convert to 25OHD in the liver - and
the liver may not be functioning well. So high dose oral D3 -
such as 50,000 or 100,000IU, ideally with a meal including fats - when
they arrive in hospital, is one way to start. If you can do oral
of IV 25OHD (calcifediol = Rayaldee
then this would be faster. Most immune system cells require
plasma 25OHD for their internal synthesis of 1,25OHD to activate their
internal (intracrine) vitamin D receptor signaling. So the whole
of immune regulation depends on a good 25OHD level.
Other cells, such as endothelial cells, require 1,25OHD calcitriol at
their membrane bound vitamin D receptors, and so may not rely directly
on plasma 25OHD levels.
Please see this recent review article:
Adequate vitamin D activation of endothelial cells achieves numerous functions:
- Increase vasodilation by releasing NO.
- Bring more calcium into the cell and release it from internal
stores to activate eNOS which produces the NO and stop AG2 catabolizing
L-arginine so it can be used by eNOS.
NO is a primary vasoactive substance that works as a potent vasodilator in addition to other vasoprotective properties such as protection from vessel inﬂammation and lesion formation.
NO acts on adjacent vascular smooth muscle cells in a paracrine manner
and induces vascular muscle relaxation . . .
. . . protects the vessel from developing atherosclerosis by inhibiting platelet adherence and aggregation, and leukocyte activation.
The trinity of COVID-19: immunity, inflammation and intervention
Here is an excellent paper, from
researchers in Singapore and Liverpool regarding the role of
inappropriate, over-inflammatory, immune responses in severe symptoms
There's no mention of vitamin D - though deficiency in this clearly
drives weakened and dysregulated immune responses which allow disease
progression and which, as this article documents, drive a lot of the
damage which occurs. Nor is there any mention of coagulation or
vasoconstriction - both key elements in the material above, which was
developed after this late April article would have been finalised.
. . . aggressive inflammatory responses
strongly implicated in the resulting damage to the airways.
Therefore, disease severity in patients is due to not only the viral
infection but also the host response.
In addition, the vast release of cytokines
by the immune system in response to the viral infection and/or
secondary infections can result in a cytokine storm and symptoms of
sepsis that are the cause of death in 28% of fatal COVID-19 cases. In
these cases, uncontrolled inflammation inflicts multi-organ damage leading to organ failure, especially of the cardiac, hepatic and renal systems.
Pyroptosis is a highly inflammatory form of programmed cell death
that is commonly seen with cytopathic viruses. A wave of local
inflammation ensues, involving increased secretion of the pro-
inflammatory cytokines and chemokines IL-6, IFNγ, MCP1 and IP-10 into
the blood of afflicted patients.
Notably, there exists a highly inflammatory monocyte-derived FCN1+
macrophage population in the bronchoalveolar lavage fluid of patients
with severe but not mild COVID-19. Also, patients with severe
disease show a significantly higher percentage of CD14+CD16+
inflammatory monocytes in peripheral blood than patients with mild
disease. These cells secrete inflammatory cytokines that contribute to
the cytokine storm, including MCP1, IP-10 and MIP1α.
Unrestrained inflammatory cell infiltration
can itself mediate damage in the lung through excessive secretion of
proteases and reactive oxygen species, in addition to the direct damage
resulting from the virus. Notably, virus was found in T
lymphocytes, macrophages and monocyte-derived dendritic cells. Direct virus killing of lymphocytes could contribute to the observed lymphopenia in patients.
Viral infection in immune cells such as monocytes and macrophages can result in aberrant cytokine production, even if viral infection is not productive. The degree to which SARS-CoV-2 targets these cells remains poorly defined.
Understanding the precise drivers of immune dysfunction is crucial to
guide the application of appropriate immunomodulatory treatments.
Indeed. The pages you are reading explain most or all of this
immune system dysfunction in terms of individual genetic variation,
lack of helminths, some dietary excesses and some very common
nutritional deficiencies - especially vitamin D, boron, omega-3 fatty
acids, vitamin C and potassium.
COVID-19: the vasculature unleashed
Here's another article concerning
endothelial failure in the lungs, with the researchers not knowing why
this happens to some people.
I wonder who created this beautiful illustration.
During homeostasis, the endothelium, surrounded by mural cells (pericytes [W]), maintains vascular integrity and barrier function. It prevents inflammation by limiting EC - immune (EC = Endothelial Cell) cell and EC - platelet interactions and inhibits coagulation by expressing coagulation inhibitors and blood clot-lysing enzymes and producing a glycocalyx (a protective layer of glycoproteins and glycolipids) with anti-coagulation properties.
Interestingly, recent studies using single-cell transcriptomics revealed endothelial
phenotypes that exhibit immunomodulatory transcriptomic signatures
typical for leukocyte recruitment, cytokine production, antigen
presentation and even scavenger activity. Compared with ECs from
other organs, lung ECs are enriched in transcriptomic signatures
indicating immunoregulation, and a subtype of lung capillary ECs
expresses high levels of genes involved in MHC class II-mediated
antigen processing, loading and presentation.
After the initial phase of viral infection, ~30% of hospitalized patients with COVID-19 develop severe disease with progressive lung damage, in part owing to an overreacting inflammatory response.
These would be mainly the patients with
the weakest and most dysregulated immune systems - the primary,
currently known (I suspect boron deficiency as well) preventable cause
of which is vitamin D deficiency. Yet vitamin D is not mentioned
in this article.
Mechanistically, the pulmonary complications result from a vascular barrier breach, leading to tissue oedema (causing lungs to build up fluid), endotheliitis, activation of coagulation pathways with potential development of disseminated intravascular coagulation (DIC) and deregulated inflammatory cell infiltration.
Reduced ACE2 activity indirectly activates the kallikrein–bradykinin pathway, increasing vascular permeability.
Immune cells, inflammatory cytokines and vasoactive molecules lead to enhanced EC contractility and the loosening of inter-endothelial junctions. In turn, this pulls ECs apart, leading to inter-endothelial gaps. Finally, the cytokines IL-1β and TNF activate glucuronidases that degrade the glycocalyx
but also upregulate hyaluronic acid synthase, leading to increased
deposition of hyaluronic acid in the extracellular matrix and promoting
fluid retention. Together, these mechanisms lead to increased vascular permeability and vascular leakage.
Vascular integrity and EC death leads to exposure of the thrombogenic basement membrane and results in the activation of the clotting cascade.
"Finally" just refers to this initial
stage of the zombie apocalypse which overcomes all these cell types in
the lungs. Note that in the description so far, and in what
follows, there is little or no mention of viral activity. Most
people deal with the virus pretty well, and things never get as bad as
just described. If you have read this page and most of the ../cv19/
page you will be at least roughly aware of the protective, largely vitamin D dependent
mechanisms in these cells which protect them from damage by pathogens,
and disallow the storm of destruction described so well here, which is
primarily driven by a weakened and dysregulated immune response.
ECs activated by IL-1β and TNF initiate coagulation by expressing
P-selectin, von Willebrand factor and fibrinogen, to which platelets bind.
In turn, ECs release trophic cytokines that further augment platelet
production. Platelets also release VEGF, which triggers ECs to
upregulate the expression of tissue factor, the prime activator of the coagulation cascade, which is also expressed by activated pericytes. In response, the body mounts countermeasures to dissolve fibrin-rich blood clots, explaining why high levels of fibrin breakdown products (D-dimers) are predictive of poor patient outcome. As a result of the DIC and clogging/congestion of the small capillaries by inflammatory cells, as well as possible thrombosis in larger vessels, lung tissue ischaemia develops, which triggers angiogenesis and potential EC hyperplasia. While the latter can aggravate ischaemia, angiogenesis can be a rescue mechanism to minimize ischaemia. However, the newly formed vessels can also promote inflammation by acting as conduits for inflammatory cells that are attracted by activated ECs.
We're not done yet . . .
Many patients with severe COVID-19 show signs of a cytokine storm. The high levels of cytokines amplify the destructive process by leading to further EC dysfunction, DIC, inflammation and vasodilation of the pulmonary capillary bed.
This explanation differs from that of Farid Jalali's #2020-Jalali-b
above - in which angiotensin II levels force vasoconstriction.
The above words seem to indicate that the unspecified mechanisms of
vasodilation (I guess nitric oxide, as noted above) prevail.
This results in
alveolar dysfunction, ARDS with hypoxic respiratory failure and
ultimately multi-organ failure and death.
EC dysfunction and
activation likely co-determine this uncontrolled immune response.
Yes, but why? This is where
anyone who has read the research I have read - and which you will read
if you follow my links - thinks of the low 25OHD levels of these
patients, and how this will reduce the ability for numerous types of
immune cell (and it seems we should count some or all endothelial cells
as immune cells too) to act strongly and successfully against the
virus, while regulating the destructive responses which cause almost
all the harm described in this article.
is because ECs promote inflammation by expressing leukocyte adhesion
molecules, thereby facilitating the accumulation and extravasation of
leukocytes, including neutrophils, which enhance tissue damage. This
is because ECs promote inflammation by expressing leukocyte adhesion
molecules, thereby facilitating the accumulation and extravasation of
leukocytes, including neutrophils, which enhance tissue damage. [Neutrophils are one of the immune system's professional phagocytes. W]
Moreover, we hypothesize that denudation of the pulmonary vasculature
could lead to activation of the complement system, [W] promoting the
accumulation of neutrophils and pro-inflammatory monocytes that enhance
the cytokine storm.
As I wrote on the main page cv19/
most of the death resulting from influenza is also due to weakened,
dysregulated, immune systems, with the same primary causes (cv19/#helminthsgone
lack of helminths, individual genetic variation, dietary excesses and
nutritional deficiencies, most particularly of vitamin D.
This is based on the observation that during
influenza virus infection, pulmonary ECs induce an amplification loop,
involving interferon-producing cells and virus-infected pulmonary
epithelial cells. Moreover, ECs seem to be gatekeepers of this [destructive and quite likely deadly] immune
response, as inhibition of the sphingosine 1 phosphate receptor 1
(S1PR1) in pulmonary ECs dampens the cytokine storm in influenza
infection. This raises the question whether pulmonary ECs have a
similar function in the COVID-19 cytokine storm and
whether S1PR1 could represent a therapeutic target. Another unexplained
observation is the excessive lymphopenia in severely ill patients with
COVID-19 and whether this relates to the recruitment of lymphocytes
away from the blood by activated lung ECs.
Yes they are a therapeutic target -
along with every other type of cell in our bodies. Everyone
should run their bodies better by giving all their cells the nutrients
they require for proper operation. This starts with 40 to 60ng/ml
When they don't do this, and their levels are 30ng/ml or less, sometimes down to 10ng/ml or less (and see ../#21authors
for an article written by 21 experts who regard vitamin D deficiency as
being only below 10ng/ml) they can get by - until a virus, bacteria or
some other insult triggers this weak and dysregulated immune
response. Then they suffer sepsis, pneumonia etc. - or in the
case of COVID-19 and even worse horror story because of the way the
virus, during this self-destructive cascade, infects immune cells which
respond to the troubles by expressing ACE2 receptors, driving a
hypercoagulative storm which wreaks likely permanent harm all over the
When people get to hospital, in these conditions, the first thing
doctors should do is try to raise their 25OHD levels to something
healthy, like 40ng/ml. Perhaps it will be too late, but I am
exceedingly perplexed, spending these months knowing how low 25OHD is
not being treated, and is condemning hundreds of thousands of people to
terrible suffering, lasting harm and death.
We should remember that the wholesale 1kg ex-factory cost of D3 for 4000IU a day is US 9 cents per year.
All these people are boron deficient too. Boron's half life is 20
hours or so, and they start of boron deficient in ordinary life,
getting about 1mg or so a day, and become even more deficient in
hospitals since doctors don't even recognise it as a nutrient.
Yet boron has numerous anti-inflammatory properties, similar to vitamin
D, but also in some ways unique to itself.
100mg of borax a day gives 11.4mg boron a day, which is surely
sufficient for repletion. This is 36 grams of borax a year, which
costs (retail, USD$10/kg) US 36 cents per year.
circumstantial evidence suggests a link between ECs, pericytes and
COVID-19. First, risk factors for COVID-19 (old age, obesity,
hypertension and diabetes mellitus) are all characterized by
pre-existing vascular dysfunction with altered EC metabolism. As
hijacking of the host metabolism is essential for virus replication and
propagation, an outstanding question is whether EC subtypes or other
vascular cells in specific pathological conditions have a metabolic
phenotype that is more attractive to SARS-CoV-2.
Old age - in the absence of 4000IU or more D3 a day supplementations -
is strongly correlated with even lower 25OHD levels than is normal
middle age. These risk factors are caused, in large part, by
vitamin D and other deficiencies, the lack of helminths etc. and
excessive dietary fats, sugars and other carbohydrates, and frequently
lack of proper exercise.
So even if the actual conditions were not risk factors in themselves
(and they are, especially obesity) then the correlation between these
conditions and risk for serious COVID-19 symptoms would be to a large
extent explained by a lifetime of inadequate vitamin D.
occasional clinical reports suggest an increased incidence of Kawasaki
disease, a vasculitis, in young children with COVID-19.
I had never heard of Kawasaki disease. It took me less than a
minute with Google Scholar, searching for it with "vitamin D" to find
the article I link to here ../#2015-Stagi
which shows that the only children who suffer and die from this
terrible condition have exceedingly low (rickets levels) of 25OHD.
Other articles on COVID-19 endothelial pathology
There are no doubt other articles with detailed accounts of COVID-19
pathology in the epithelium of the lungs. I don't have time to
find or read them all, but here are some of potential interest:
Vitamin K depletion in severe COVID-19 contributing to coagulation?
I haven't read this yet. Would
vitamin K supplementation reduce the most destructive aspect of severe
COVID-19: the hypercoagulative state?
MATH+ Protocol for early intervention to reduce cytokine storm
and so reduce or eliminate the deadly hypercoagulative state
The website of the Front
Line COVID-19 Critical Care Alliance
Line COVID-19 Critical Care Working Group covid19criticalcare.com
and before that: recoverywithoutwalls.com/covidprotocol/ .)
Please follow all the links to protocol, press release and other
documents there, as well as documents linked from theirs:
in Critical Illness: Glucocorticoid Receptor-alpha Master Regulator of
Meduri & Chrousos
A 2018 article about this by Paul Marik and colleagues: Ascorbic
acid, corticosteroids, and thiamine in sepsis: a review of the biologic
rationale and the present state of clinical evaluation www.ncbi.nlm.nih.gov/pmc/articles/PMC6206928/
contains a big diagram and explanation.
The protocol concerns the early
use of methylprednisolone, intravenous vitamin C, thiamine (), low
molecular weight heparin and other compounds, including vitamin D and
melatonin to reduce or prevent the development of
the cytokine storm which causes the hypercoagulative state which is
what harms and kills people. Other elements are nasal cannula
oxygen with prone positioning and avoidance of incubation as much as
The Marik Protocol
Paul Marik MD is a member of the FLCCC Alliance mentioned above. His work predates COVID-19 and is applicable
to most ICU patients, especially those with sepsis and lung injury. His protocol is available from:
Paul Marik's approach was discussed in March 2017 at https://emcrit.org/pulmcrit/metabolic-sepsis-resuscitation/
. At that page, the second video is of some nurses at
the East Virginia Medical School hospital. At 1:09, Team
Coordinator Kathi Hudkins recalls her team's initial thoughts on
Dr Marik's protocols:
"This had to be some soft of fallacy. It just seemed too
But then their patients started getting better.
Despite the obvious necessity of supplementing a micronutrient
which is essential to immune function, Paul Marik faces an uphill
battle having his ideas accepted - and he considers it unethical
to do an RCT, since half his patients would be denied a treatment
which he knows works.Dr Paul Marik has written over 400 peer-reviewed journal
articles, 50 book chapters and 4 critical care books.
In 2017 criticalcarereviews.com/...cure-for-sepsis-with-paul-marik he said:
colleagues, when I told them what was going on, thought it was the
biggest load of nonsense they had ever seen. But then they
actually saw that none of our patients were dying. And then our
CEO saw that none of the patients were dying. So this has become
instituted through the whole healthcare system.
"We were going to do a randomised clinical trial, but we couldn't -
because it would have been unethical."
At this page there is also a 2020-05-28 video - which is actually at YouTube
- in which Professor Marik discusses COVID-19 in general and how he and
his team treat it. His part in the video starts at 45
At 50:30 he mentions vitamin D status as one of the factors affecting
COVID-19 outcomes - and the effect of high geographical latitude on low
vitamin D levels. In US states north of 40° there is a much
higher risk of COVID-19 mortality.
COVID-19 results in a very high expression of inflammatory
cytokines, chemokines, IL-6 and IL-1 [much more so than common
respiratory viruses, including influenza].
What makes this virus so unique and so smart is that while it
upregulates the inflammatory response, it downregulates the expression
Interferon type 1 and type 3 are the major host defenses against viral
infections. COVID-19, as opposed to influenza, results in a
limited antiviral response. So there is a marked imbalance between the
host immune response in getting rid of the virus and this profound inflammatory response.
EMCrit discussion on timing of corticosteroids
Josh Farkas and
commenters discuss the timing and strength of corticosteroid use with
Dr Cameron Kyle-Sidell's videos on how different COVID-19 is from
what he trained for
L. Gattinoni et al. on L and H "phenotypes"
Dr Farid Jalali's COVID-19 diagram concerning cytokine storm and
resulting hypercoagulative state
Farid Jalali https://twitter.com/farid__jalali
tweeted a detailed diagram https://twitter.com/farid__jalali/status/1247036001349849088
depicting the cytokine storm turning a moderately PAI-1 elevated
coagulative state, which apparently can be controlled with heparin,
into a hypercoagulable Progressive Thrombotic Cascade. The long
progression of this thrombosis apparently enables the oxygen levels to
drop slowly enough that the patient copes with it better than would be
the case with normal ARDS. This leads to microvascular thrombosis
in the lungs, heart, kidney, gut, pancreas, skin and CNS - and so
frequently to death.
He states that COVID-19 is cytopathic to the endothelium and that
this degenerates (as far as I know, in the absence of vitamin C etc.
treatment) into the Severe COVID-19 cytokine storm. This makes me
think that the vitamin C, corticosteroid etc. treatment prevents the
virus and the consequent (at least in these patients) immune response
doing so much harm to the endothelium.
../ to the main COVID-19
page of this site.
To the index page of this site: ../../
Contact details and copyright information: ../../contact/
2020 Robin Whittle - please link to this site rather than copy the
whole of its contents Daylesford, Victoria,