Fever curves are an interesting source of amusement for those that have the ability to collect the data.  This is especially true for those experiencing periodic fever or chills.

Reducing Fevers:  propacetamol (Tylenol) versus  dexibuprofen

Paracetamol, acetominophen, propacetamol are inhibitors of cyclooxygenase  (Cox).  Some references suggest it might interact with the endocannabinoid system.     Dexibuprofen, like its more structurally heterogenous ibuprofen,  is a nonsteroidal anti-inflammatory drug (NSAID).  Cox is part of the pathway that converts arachindonic acid (AA) to the pro-inflammatory cytokine PGE2.

Fevers in kids [1]

This study looked at fevers that accompany upper respiratory track infections (URTI) in children.  The kids were given oral dexibuprofen or intravenous propacetamol.  The rationale was that if a kid is vomiting, oral antipyretics are ineffective.   Auxillary (armpit) temperatures were recorded every 30 minutes.  This was an Equivalence Trial, a trial with the primary objective of showing that the response to intravenous propacetamol differs from oral Ibuprofen by an amount which is clinically unimportant. This is usually demonstrated by showing that the true treatment difference is likely to lie between a lower and an upper equivalence margin of clinically acceptable differences.

Inclusion criteria

• Patients ranging in age from 6 months to 14 years admitted for URTI and presenting with fever (defined as body temperature of the axillar fossa ≥38.0 °C) at the time of admission were included.
• URTI was diagnosed based on disease history and physical examination carried out by the attending pediatricians.

Exclusion criteria

• Patients were excluded under the following circumstances: the patient had been administered antipyretics within 4 h prior to admission,
• a history of febrile crisis within the past 6 months,
• the presence of severe hematological abnormality, currently receiving treated for or was treated within the past 6 months for nephrologic, hepatologic, pulmonary, endocrine, hematologic, or cardiologic illnesses, neurologic or central nervous system abnormality,
• diabetes currently not under control,
• suspected lower respiratory tract infection,
• severe hemolytic anemia,
• under maintenance therapy for bronchial asthma, asthma, urticarial,
• allergic reaction history when using aspirin or NSAIDs, physical or psychological status deemed inappropriate for a clinical trial,
• participation in another clinical trial involving other drug(s) within the past 4 weeks, and failure to receive informed consent from the patient or parent.

This is what a temperature curve looks like.  An attempt has been made to shade the area under the Dexibuprofen  curve (gray) versus the Propanecetamol (blue).  Notethat the X-axis is time and the Y-axis temperature.  The larger the area, the more time the child spends feverish.  Some studies on ClinicalTrials.gov like to use  37.0 °C for calculating area under the curve (AUC).

Outcome measurements

• The primary outcome measurement was the difference in body temperature reduction at 4 h after antipyretic administration between the study and control groups.

The authors failed to demonstrate superiority of intravenous propacetamol oral Dexibuprofen.

• The secondary efficacy variables were range of body temperature reduction at 4 h after antipyretic administration, the Area Under the Curve (AUC) of body temperature change until 6 h after antipyretic administration-and-time relationship,
• the maximum value of body temperature reduction within the 6 h after antipyretic administration,
• the number of patients whose body temperature normalized (< 37.0 °C) at 6 h after antipyretic administration
• the time point when body temperature first reached< 37.0 °C.

In stroke patients [2]

Seventy-five patients with acute ischemic stroke confined to the anterior circulation were randomized to 6 times daily treatments during 5 days after the stroke  with either

• 1000 mg acetaminophen
• 400 mg ibuprofen
• placebo

This study used tympanic temperatures measured every two hours.  Treatment was started within 24 hours from the onset of symptoms. Body temperatures were measured at 2-hour intervals during the first 24 hours, and at 6-hour intervals thereafter.  We don’t know how diurnal variations in body temperature came into play.  We can hypothesize that these patients were not getting a lot of natural sunlight to set their circadian rhythms into motion.

Outcome Measures

Clinical trials have primary and secondary measures to see if the tested intervention has the desired effect on the patient’s outcome.
The primary outcome was body temperature measured rectally at 24 hours from start of treatment. Secondary outcomes were change from baseline temperature at 1 and 5 days from start of treatment, and time with elevated body temperature (> 37.0°C) (area under the curve) during the first 24 hours and the first five days.

Patients could be included if they had

• acute ischemic stroke in the anterior circulation
• a body temperature between 36.0°C and 39.0°C,
• computed tomography (CT) scan that was compatible with acute ischemic stroke
• a focal deficit without rapid improvement
• and a possibility to start treatment within 24 h after stroke onset.

Exclusion

• Patients with a posterior circulation stroke were not included because occasional patients could have severe disturbances of temperature regulation through involvement of the hypothalamus.

Results…

Without intervention, the tympanic temperature increases about 0.4 °C  in first 8-10 hours after recording, which places us at 8 to 34 hours after the stroke. After about 24 hours the temperature levels off to about 0.2 °C  above the starting point.

The Barthel Index is a measure of how much a patient needs help with aspects of daily life such as bathing and using the toilet.  A score of 20 is functional independence.  TheModified Rankin Score is also measures the functional impact of a stroke  A score of 2 indicates not back to normal but able to live independently.  A score of 5 indicates bedridden.

1. The antipyretic efficacy and safety of propacetamol compared with dexibuprofen in febrile children: a multicenter, randomized, double-blind, comparative, phase 3 clinical trial
   Seung Jun Choi, Sena Moon, Ui Yoon Choi, Yoon Hong Chun, Jung Hyun Lee, Jung Woo Rhim, Jin Lee, Hwang Min Kim, Dae Chul Jeong BMC Pediatr. 2018; 18: 201. [PMC free article]
2. Dippel, Diederik W J et al. “Effect of paracetamol (acetaminophen) and ibuprofen on body temperature in acute ischemic stroke PISA, a phase II double-blind, randomized, placebo-controlled trial [ISRCTN98608690].” BMC cardiovascular disorders vol. 3 (2003): 2. doi:10.1186/1471-2261-3-2 [PMC free article]

 Medical foods do not require a prescription and are not regulated as drugs.  They may provide nutrition when the patient is too incapacitated to get said nutrition from a normal diet.  In this case we have a sick guy who just doesn’t feel like eating copper rich foods like liver and kale.  In this post we will cover how copper may play a role in the renin angiotensin system (RAS) that may be compromised in severe Covid-19 and other infections. 

What is the Renin Angiotensin System (RAS)?

Qin and coworkers (2020) first noted hypertension as a risk factor for severe versus mild forms of COVID-19.  Lippi and coauthors (March 31, 2020) summarized literature of numerous studies and found a consistent correlation between severe COVID-19 and hypertension.  Which came first?  Hypertension or Severe Covid-19?  Was hypertension pre-existing or the result of the infection?  Was pre-existing hypertension controlled by the use of an ACE inhibitor that might have increased the expression of the ACE2 enzyme/COVID-19 receptor?  Or was pre-existing hypertension controlled by use of  β-adrenergic receptor blockers, controlled by life style changes like low sodium diets, or left uncontrolled? Hao Cheng and colleagues of the Peking University First Hospital of Beijing reviewed risk factors or severe COVID-19 infections and the COVID-19 receptor angiotensin converting enzyme 2 (ACE2). ACE2 is a receptor for other corona viruses.  Let us take a closer look at the RAS.

 When blood flow to the kidney is decreased, plasma pro-renin is cleaved to the active protease renin. Renin cleaves angiotensinogen to angtiotensin I. Angiotensin converting enzyme (ACE) cleaves angiotensin I to angiotensin II. Angiotensin II binds to the angiotensin receptor 1 (AT1). The primary result is increased expression of NADPH oxidase, a super oxide generating enzyme. Superoxide reacts with with the vasodilator nitric oxide (NO) to produce peroxynitrite. Blood vessels constrict and renal blood flow is increased.

Cu/Zn SOD3 and RAS, a closer look

ACE2, aka the Covid-19 receptor, used to be primarily regarded as the first brake to the RAS.  Ozumi (2012) discovered a second brake: increased expression of Cu/Zn superoxide dismuase 3 (SOD3). Activation of the AT1 receptor might even stimulate Cu(I) uptake by the copper transporter Ctr1. Cu(I) is handed off to the Cu(I) chaperone Atox1, a transcription factor for the SOD3 gene. 

Multiple levels of regulation…

• Sean Didion (2012) chose not to follow up on the Ozumi (2012) suggestion that ATR1 modulates the Ctr1 channel.  Didion focused on demonstrating that angiotensin II also increases protein levels of Atox1 and SOD3.
• Meanwhile, Wang (2016) reported that SOD3 levels are decreased in the brains of ACE2 knock out mice. If anything, angiotensin II should increase SOD3 in these mice. It should be noted that both the MAS and AT1 receptors signal via heterotrimeric G-proteins.
• Abouhashem (May 2020) isolated single cell RNA from alveolar type II epithelial cells.  These cells came from elderly and young donors.  The mRNA were sequenced and expression levels compared.   The rationale was taht colocalization of ACE2 and TMPRSS2 enables Covid-19 to invade cells. Expression levels of these genes in the alveolar type II cells of elderly and young patients were comparable.  In cells from the elderly, 263 genes were down regulated and 95 up regulated.   Superoxide dismutase 3 (SOD3) was identified as the top-ranked gene that was most down regulated in the elderly.  The authors proposed that SOD3 and the ATF4 transcription factor -related antioxidants will work in synergy with promising antiviral drugs such as remdesivir to further improve COVID-19 outcomes in the elderly.
• Cheng (2020) discussed ACE2 as one means of applying the brake to the RAS increase in blood pressure. COVID-19 binding to ACE2 might compromise this brake. There is an internal feedback loop that involves Cu(I). One way or another, SOD3 expression is increased by angiotensin 1-7 and aginiotensin II (aka 1-8). A functional SOD3 enzyme requires repletion with copper in the +1 oxidation state.

In summary

To the invited guest reading this post, we are not making claims at this point.  We do want to remind you that the copper binding protein Atox1 regulates the expression of SOD3.  We do not know if these elderly volunteers were copper deficient compared to younger volunteers.

RAS, not just corona virus infections

The COVID-19 infection is just one example in which the RAS system might be activated: (1) decreased fluid intake due to compromised GI function that is not quite diarrhea and (2) direct activity of the virus on the RAS. Naturally healthcare professionals would be involved in assays involved with plasma SOD3 activity and copper content of ceruloplasmin.

But also bacterial infections

Bacterial sepsis involves SOD3. Constantino (2014) induced bacterial sepsis in a rat cecal ligation and preforation model. Cecal perforation would be akin to an appendicitis in humans. These authors found an increase in SOD3 levels following sepsis that were not able to totally prevent oxidative stress and inflammation associated with ONOO− production. While the rats should not have been copper deficient, the role of supplemental to replete the new production of SOD3 was not examined. Mechanism(s) of increased SOD3 were not examined, but one would expect activation of the RAS in response to the massive vascular collapse that is associated with sepsis. Human patients experiencing an appendicitis would not be boosting their serum copper via normal Cu(I) found in foods. Cu(I)NA₂ administered as a medical food would have to be done under the supervision of a physician.

And also yeast infections

Candida (yeast ) infections offer another twist to SOD3 and Cu(I). Yeast express a zinc free, Cu only, superoxide dismuase (SOD5) that enables them to survive super oxide produced in the phagosomes of macrophage and neutrophils. SOD5 is an extracellular enzymes attached to the cell membrane by way of glyosylphosphatidylinositol linkages (Gleson 3015).

Unlike mammalian secreted SOD3, SOD5 does not use these Golgi proteins

• the copper chaperone Atox1
• copper-transporting ATPases

Gleason (2014) also proved that,  unlike intracellular mammalian SOD1, yeast SOD5 does not require

• a CCS chaperone
• Yeast acquires Cu(II) from the extracellular mileau of the host.

This  host source might be Cu(II) bound to albumin. Robinette (2020) compared the affinities yeast copper only SOD5 and bovine SOD1 for copper using Cu(II) chelators. They found that SOD1 has a higher affinity for Cu(II) than yeast SOD5. The right Cu(I) copper supplement may

1. feed the patient’s SOD3 and SOD1
2. while not feeding Cu(II) to SOD5 of the yeast pathogen.

If one cannot absorb copper from food because of an illness

. It is proposed that Cu(I)NA₂ is a way of obtaining copper in its +1 oxidation state for those who are too sick to eat the large amounts of copper rich foods to get said copper in its natural +1 oxidation state. Any infection, be it fungal, viral, or bacterial is associated with large production of the reactive oxygen species super oxide. 

Septic shock is also associated with vascular collapse and the associated activation of the Renin-Angiosystem-System.  Angiotnsin II has even been explored as a means of preventing vascular collapse and multi-organ failure (Corrêa  2015). 

As reviewed in this post, angiotensin II is also a way of boosting blood levels of Cu/Zn SOD3.  

No appetite or unable to eat copper containing foods

While Cu(I)NA₂ has the same oxidation state as copper naturally occurring in the food we eat, it is a processed product to deliver required amounts of copper to patients who are too ill to large amounts of food. In the case of infections it is critical that enzymes such as superoxide dismutase are replete with the copper cofactor.

Feeling too sick to eat is a common symptom of many illnesses, not just infections. COVID-19’s affect on the GI tract may be under recognized according to a letter by Weicheng Liang and colleagues to the British Medical Journal Gut. These authors argued that while only a smaller fraction of COVID-19 patients had out right diarrhea, the small intestine might be compromised and the sight of COVID-19 infection due to the extensive GI expression of ACE2, the receptor for the COVID-19 spike glycoprotein.

Unable to eat

The appendicitis patient may be NPO, “nil per os”…. “nothing by mouth.”    A few arguments for non oral routes for drug status…

1. No duodenum…  Griffith (2009) was one of the first to report copper deficiency secondary to the “roux-en-Y” gastric bypass that may takes the duodenum and 100-300 cm of the proximal jejunum. This is one of many papers addressing this particular problem.
2. Absorption by the colon  Charlie Barker, owner and CEO of Mitosynergy, has talked of colonic andministration of Cu(I)NA₂ via the rectal route. My big questions for gatroenterlogists would  (1) Are appendicitis patients NPO because Vagas nerve stimulation make the condition worse?  (2) Does injecting something into the patient’s colon via the rectum stimulate the Vagus nerve?
3.  Cu+ transporters in the colon Here is an overview of the Cu(I) transporter Ctr1 offered by ProteinAtlas.org.  It become readily apparent from the brown antibody staining why gastric bypass patients may become copper deficient.

Cu(I)NA₂ already has clearance from the FDA as a new dietary ingredient. In an infection such as COVID-19 that can compromise the gastrointestinal tract, obtaining the proper dose may require medical supervision. This supervision may include

• rectal administration bypassing the oral route
• measuring the superoxide dismutase level in the patient’s blood
• or the copper load in the Cu(I) carrier protein ceruloplasmin.

Additional information

• Other routes of entry have been covered in cuprous niacin as a drug  .
• Cu(I)NA₂‘s role in boosting immune function that is separate from its role in Atox1 mediated transcription of SOD3 and as a cofactor in Cu/Zn SOD3.
• We have fish and human studies for immune system boosting.

References

Cheng H, Wang Y, Wang GQ. (2020) Organ-protective Effect of Angiotensin-converting Enzyme 2 and its Effect on the Prognosis of COVID-19. J Med Virol. 2020 Mar 27. Review. Link

Constantino L, Gonçalves RC, Giombelli VR, Tomasi CD, Vuolo F, Kist LW, de Oliveira GM, Pasquali MA, Bogo MR, Mauad T, Horn A Jr, Melo KV, Fernandes C, Moreira JC, Ritter C, Dal-Pizzol F. (2014) Regulation of lung oxidative damage by endogenous superoxide dismutase in sepsis. Intensive Care Med Exp. 2014 Dec;2(1):17. Link

Corrêa TD, Takala J, Jakob SM.(2015)Angiotensin II in septic shock. Crit Care. 2015 Mar 16;19:98 Link

Didion SP.(2012) Antioxidant 1 in hypertension: more than just a copper chaperone. Hypertension. 60(2):285-7 Link

Gleason JE, Galaleldeen A, Peterson RL, Taylor AB, Holloway SP, Waninger-Saroni J, Cormack BP, Cabelli DE, Hart PJ, Culotta VC. (2014) Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense Proc Natl Acad Sci U S A. 111(16):5866-71. Link

Griffith DP, Liff DA, Ziegler TR, Esper GJ, Winton EF.(2009) Acquired copper deficiency: a potentially serious and preventable complication following gastric bypass surgery. Obesity (Silver Spring).17(4):827-31. Link

Liang W, Feng Z, Rao S, Xiao C, Xue X, Lin Z, Zhang Q, Qi W. (2020) Diarrhoea may be underestimated: a missing link in 2019 novel coronavirus. Gut. 2020 Feb 26. pii: gutjnl-2020-320832. Link

Lippi G, Wong J, Henry BM.(2020) Hypertension and its severity or mortality in Coronavirus Disease 2019 (COVID-19): a pooled analysis. Pol Arch Intern Med. 2020 Mar 31. Link

Meng J, Xiao G, Zhang J, He X, Ou M, Bi J, Yang R, Di W, Wang Z, Li Z, Gao H, Liu L, Zhang G. (2020)Renin-angiotensin system inhibitors improve the clinical outcomes of COVID-19 patients with hypertension. Emerg Microbes Infect. 9(1):757-760. Link

Ozumi K, Sudhahar V, Kim HW, Chen GF, Kohno T, Finney L, Vogt S, McKinney RD, Ushio-Fukai M, Fukai T. (2012) Role of copper transport protein antioxidant 1 in angiotensin II-induced hypertension: a key regulator of extracellular superoxide dismutase.Hypertension. 60(2):476-86. Link

Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang W, Tian DS. (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin Infect Dis. 2020 Mar 12. pii: ciaa248.

Robinett NG, Culbertson EM, Peterson RL, Sanchez H, Andes DR, Nett JE, Culotta VC. (2019) Exploiting the vulnerable active site of a copper-only superoxide dismutase to disrupt fungal pathogenesis. J Biol Chem. 294(8):2700-2713 Link

Wang XL, Iwanami J, Min LJ, Tsukuda K, Nakaoka H, Bai HY, Shan BS, Kan-No H, Kukida M, Chisaka T, Yamauchi T, Higaki A, Mogi M, Horiuchi M. (2016) Deficiency of angiotensin-converting enzyme 2 causes deterioration of cognitive function. NPJ Aging Mech Dis. 2016 Oct 20;2:16024. Link

These are some thoughts addressing whether Cu(I)NA₂  could be used as a medical food to treat various infections in addition to COVID-19 that is making the news as of this posting.   This “proper copper thought was inspired by the study of Qin (2020) study that showed an increase in the neutrophil to lymphocyte ratio in severe cases of COVID-19.   Early studies of  Cu(I)NA₂  show that it decreases neutrophils and increases lymphocytes in humans and fish.  The ratio of neutrophils to lymphocytes has gained popularity as a prognostic marker in infectious diseases.   Don’t we need both?  The following is a Devil’s Advocate examination of copper and its deficiency in sepsis.  Your comments are invited.

While the pandemic is over, we still have SARS-Cov2 and plenty of other infections.

Does the neutrophil to lymphocyte ratio matter?

Dr Lagunas-Ramal (2020) has called for use of the neutrophil to lymphocyte ratio as a metric.   of hyper-inflammation to predict which cases of COVID-19 are likely to progress to becoming severe.  Most hospitals in places like Mexico have the instrumentation to perform such tests.  Cu(I)NA₂ has been shown to decrease neutrophils and increase lymphocytes in a human clinical trial and in two fish studies. Neutrophils are not only the first line of defense of the innate immune system but also a source of reactive oxygen species (Kircher 2012).

Neutrophils have gotten a bad reputation in the prognostic value of the neutrophil to lymphocyte ratio. Kirchner and coworkers (2012) demonstrated that NADPH oxidase generated superoxide and myeloperoxidase generated hypochlous (bleach) are involved in production of the neutrophil extracellular trap (NET). Myeloperoxidase produces hypochlorous acid from the Cl^– anion and hydrogen peroxided, H₂O₂. NET is composed of DNA and a variety of antimicrobial proteins.

As of 15 April 2020 (Velavan review) low lymphocyte counts were still considered to be a predictor of severe COVID-19 cases.  T- and B-cell differentiation factor  interleukin 6 (IL-6), C-reactive protein (CRP), and fimbrin degradation product D-dimer were added to the list of predictors of severe COVID-19.

Do NETs catch viruses?

Do NETs catch viruses? We do not currently have information on the Corona virus family but have the influenza study of Tang (2019).

These authors started with 720 patients who met the World Health Organization’s criteria for influenza. Influenza cases were confirmed with PCR. Messenger RNA was extracted from whole blood from confirmed cases in order to determine which mRNA best differentiate severe cases that require a ventilator versus those that do not. Healthy controls were also added to the analysis.

• Each mRNA message was identified and quantitated with microarray analysis.
• Messages were grouped according to protein function in the disease process.
• Of the six modules with predictive power, the “neutrophil module” had the best ability to differentiate severe from moderate cases of influenza.
• The NET protein myeloperoxidase was one of the top proteins expressed more in severe cases of influenza compared to mild cases.

Ni and coworkers (2019) found that septic patients admitted to the hospital with a high neutrophil to lymphocyte ratio were less likely to die in the hospital. Similar use of the NLR has yet to take hold in the literature as a prognostic test for yeast pathogens like Candida species. A recent review (Patricio 2019) has compared differences in the immune response to Candida which may involve
outer layer of mannose rich N-linked glyco proteins
an inner layer of chitin
activation of host NADPH oxidase, an iron containing generator of superoxide
activation of NET along with enzymes that attack the fungal pathogen

The Patricio review (2019) goes into great detail of the ways in which the innate immune and adaptive immune systems communicate with one another by way of cytokines. These authors argue that if the balance shifts to the innate immune system, persistent inflammation-immune suppression, catabolism syndrome (PICS) can occur. Compensatory anti-inflammatory response syndrome (CARS) occurs when anti-inflammatory cytokines, some of which are secreted by T and B lymphocytes, turn off the immune reaction prematurely. These authors suggested the value of prognostic tests. We need innate and adaptive arms our immune systems. From early studies we know that neutrophils need copper. These early studies supplied copper in the Cu(II) oxidation state. We are proposing that copper in the Cu(I) oxidation state may bring better balance.

and COVID-19 in particular?

A review published in April of 2020 (Barnes) reported finding evidence of NET deposits in a lung biopsy of a deceased COVID-19 patient.  This prompted the authors to review the literature.  The authors made some interesting points.

• Histones in NET also bind to phospholipids in platelets triggering the clotting cascade by activating platelets.  This might explain blot clots and strokes seen in younger COVID-19 patients.
• Barnes (2020) proposed a loop that involves NET  ⇒ IL1β⇒accelerated
• Barnes (2020) made brief mention of neutrophil elastase, a protease that degrades the extracellular matrix.   This damage may be repaired by lysyl oxidase (LOX),a copper containing amine oxidase that cross links extracellular matrix proteins elastin and collagen.

Another very recent study from Yu Zuo and collaborators (2020) found myeloperoxidase-DNA complexes and citrinullated histones in the sera of COVID-19 patients.   NETs were found to be associated with COVID-19 cases requiring mechanical ventilation. Further correlation was found with NET components cell-free DNA- myeloperoxidase.    COVID-19 sera trigger control neutrophils to release NETs.

Early Studies: Neutrophil and Macrophage activity in Cu deficiency

Jones and Suttle (1981) compared the ability of leukocytes ( presumably macrophage and neutrophils) from copper deficient (< 8 µM in plasma ) and copper sufficient (> 8 µM in plasma ) ewes and lambs to kill the yeast pathogen Candida albicans. Copper deficiency in ewes and lambs decreased the candidacidal activity in ewes and lambs. Copper deficiency further resulted in a decrease in superoxide dismuase activity in erythrocytes and leukocytes.

Babu and Failla (1990) studied the affect of dietary copper on macrophage and neutrophil activity in weaned male rats for a period of five weeks.

• adequate (7 mg/kg diet; +Cu)
• deficient (0.7 mg/kg diet; -Cu)

The following were found to decrease as a result of five week copper deficiency

• Cellular Cu concentration
• activity of Cu,Zn superoxide dismutase (Cu,Zn-SOD),
• neutrophil generation of superoxide anion (O₂^–) in response to opsonized zymosan, phorbol myristate acetate
• candidacidal activity

While phagocytic activity was independent of copper, survival of Candida albicans injected into the peritoneal cavity was greater in Cu-deficient rats than in controls. The authors tested a gradient of dietary copper to differentiate between the double edge sword of superoxide to kill the yeast and SOD to prevent the super oxide from killing the rat host. Candidacidal activity were significantly lower in neutrophils from rats fed diets with less than or equal to 2.7 mg Cu/kg feed compared to control cells.
Reduced erythrocyte Cu,Zn-SOD activity was observed only when dietary Cu was less than or equal to 2.0 mg/kg feed. A follow up study (Balla 1994) noted an increase in T cell proliferation in male mice on copper sufficient versus copper deficient diets in response to a mitogen that seems to operate via an Interleukin pathway.

Of the Cu carrier protein and neutrophils

and coworkers were interested in the role of Cu(I) bound ceruloplasmin protects neutrophils from apoptosis, programmed cell death, as a means of ending the acute phase of a neturophil mediated immune reaction. Several lines of evidence led them to this interest

• Ceruloplasmin (CP) is a Cu and Fe carrier protein P is a ferro:O2-oxidoreductase.
• CP is found in the blood plasma as well as interstitial fluid.
• CP concentration increases 4x in response to inflammation.
• CP is an anionic protein that interacts with and inhibits cationic proteins of the NET such as lactoferrin and myeloperoxidase.

The authors were primarily interested in an in vitro model of chronic inflammation induced by the cytokine TNF-α . They compared Cu replete with holo CP as well as some proteolytic fragments. The Cu replete versus apo (copper deficient) are the most relevant for Cu(I)NA2 as a medical food for sepsis.

Some findings of this study are

• Cu CP, but not apo CP, reduced intracellular super oxide generation by neutrophils.
• This was observed n the presence and absence of TNFα.
• Intracellular reactive species, that include H2O2, were increased by Cu(I) CP but not by apo CP.
• Intact, Cu CP promoted neutrophil survival.

The observation that superoxide decreased and H₂O₂ increased suggested that intracellular Cu/Zn SOD1 might have been Cu deficient.

And there are more Cu-ceruloplasmin interactions…

Kostevich (2015) demonstrated that Cu is necessary for CP binding to macrophage migration inhibitory factor (MIF). MIF is considered pro-inflammatory. Increased MIF levels are correlated with low survival in sepsis patients.  This is not the end of the list.  If Cu(I)NA₂ is to be used as a medical food in patients with sepsis, perhaps a good index of dosing would be measuring Cu content of ceruloplasmin in the patients serum.  Neutropils are good first responders.  They just need to be kept in check with copper containing enzymes so that second ponderers can take over.

References

Babu U, Failla ML. Respiratory burst and candidacidal activity of peritoneal macrophages are impaired in copper-deficient rats. J Nutr. (1990) 120:1692–9. 10.1093/jn/120.12.1692

Babu U, Failla ML. (1990) Copper status and function of neutrophils are reversibly depressed in marginally and severely copper-deficient rats. J Nutr. 120:1700–9. 10.1093/jn/120.12.1700

Bala S, Failla ML, Lunney J. (1990) Alterations in Splenic Lymphoid Cell Subsets and Activation Antigens in Copper-Deficient Rats. Ann N Y Acad Sci 1990;587:283–5. Link

Barnes BJ, Adrover JM, Baxter-Stoltzfus A, Borczuk A, Cools-Lartigue J, Crawford JM, Daßler-Plenker J, Guerci P, Huynh C, Knight JS, Loda M, Looney MR, McAllister F, Rayes R, Renaud S, Rousseau S, Salvatore S, Schwartz RE, Spicer JD, Yost CC, Weber A, Zuo Y, Egeblad M. (2020) Targeting potential drivers of COVID-19: Neutrophil extracellular traps.J Exp Med. 2020 Jun 1;217(6). pii: e20200652 Link

Cintia PG, Leonardo M, Israel OR, Andrea S, Beatriz VL, Elena DM.(2016) Superoxide Dismutase Activity, Hydrogen Peroxide Steady-State Concentration, and Bactericidal and Phagocytic Activities Against Moraxella bovis, in Neutrophils Isolated from Copper-Deficient Bovines. Biol Trace Elem Res. 171(1):94-100.

Golenkina EA, Viryasova GM, Galkina SI, Gaponova TV, Sud’ina GF, Sokolov AV. (2018) Fine Regulation of Neutrophil Oxidative Status and Apoptosis by Ceruloplasmin and Its Derivatives. Cells. 2018 Jan 12;7(1). pii: E8 Link

Jones DG, Suttle NF.(1981) Some effects of copper deficiency on leucocyte function in sheep and cattle. Res Vet Sci.31(2):151-6.

Kamiya T, Takeuchi K, Fukudome S, Hara H, Adachi T. (2018) Copper chaperone antioxidant-1, Atox-1, is involved in the induction of SOD3 in THP-1 cells. Biometals.31(1):61-68.

Kirchner T, Möller S, Klinger M, Solbach W, Laskay T, Behnen M. (2012) The impact of various reactive oxygen species on the formation of neutrophil extracellular traps. Mediators Inflamm. 2012;2012:849136

Kostevich VA, Sokolov AV, Grudinina NA, Zakharova ET, Samygina VR, Vasilyev VB.(2015)Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions. Biometals. 28(5):817-26

Lagunas-Rangel FA. (2020) Neutrophil-to-Lymphocyte ratio and Lymphocyte-to-C-reactive protein ratio in patients with severe coronavirus disease 2019 (COVID-19): A meta-analysis J Med Virol. 2020 Apr 3. doi: 10.1002/jmv.25819. [Epub ahead of print]

Ni J, Wang H, Li Y, Shu Y, Liu Y. (2019) Neutrophil to lymphocyte ratio (NLR) as a prognostic marker for in-hospital mortality of patients with sepsis: A secondary analysis based on a single-center, retrospective, cohort study. Medicine (Baltimore). 98(46):e18029. Link

Patricio P, Paiva JA, Borrego LM. (2019) Immune Response in Bacterial and Candida Sepsis. Eur J Microbiol Immunol (Bp). 9(4):105-113. Link

Tang BM, Shojaei M, Teoh S, Meyers A, Ho J, Ball TB, Keynan Y, Pisipati A, Kumar A, Eisen DP, Lai K, Gillett M, Santram R, Geffers R, Schreiber J, Mozhui K, Huang S, Parnell GP, Nalos M, Holubova M, Chew T, Booth D, Kumar A, McLean A, Schughart K. (2019) Neutrophils-related host factors associated with severe disease and fatality in patients with influenza infection. Nat Commun. 31;10(1):3422. Link

Velavan TP, Meyer CG. (2020) Mild versus severe COVID-19: laboratory markers. Int J Infect Dis. 2020 Apr 25. pii: S1201-9712(20)30277-0. Link

Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang W, Tian DS (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China.Clin Infect Dis. 2020 Mar 12. pii: ciaa248 Link

Zuo Y, Yalavarthi S, Shi H, Gockman K, Zuo M, Madison JA, Blair CN, Weber A, Barnes BJ, Egeblad M, Woods RJ, Kanthi Y, Knight JS.(2020)Neutrophil extracellular traps in COVID-19. JCI Insight. 2020 Apr 24. pii: 138999 Link

This post is about some of the crazy stuff going on in trying to find better ways to treat Covid-19.  Many of the treatments proposed to be encapsulated in this nanoparticles are already losing favor.

Laboratory Investigation of Effectiveness of Microencapsulated Biocontrol Antagonist Therapy and Immunogenicity Treatment for COVID-19 Virus

Alternate Title:

Investigation of Enhanced Experimental Nasal Muscosa Biocontrol Therapy and Immunogenicity Treatment for MERS, SARS, COV-2, (Corona-19), COVID-19 Viruses Using Nanoscopic Biopolymer/Biometal Micro-NanoCapsules With CuI Copper(I) iodide, Neocuproine Copper(II), Bathocuproine Disulfonic Acid Copper(I), Chloroquine, CBD and ACE-2 Inhibitors as Combined Functioning Antagonists

What is still not clear is if the core contents are actually part of the CuI matrix on the outside.  Many of the proposed delivery drugs have nitrogens that may form complexes with cuprous and cupric copper.  Many of the proposed contents are losing favor for the treatment of Covid-19.

Medications used to treat parasitic infections

Melarsoprol is an acute toxin  that is  under investigation in clinical trial NCT00330148 (Randomized Clinical Trial of Three Drug Combinations for Late-Stage Gambiense Human African Trypanosomiasis).  This compound is very toxic to aquatic life with long lasting effects [Warning Hazardous to the aquatic environment, long-term hazard]   While melarsoprol is used to treat Trypanosoma brucei, the causative agent of sleeping sickness, it only worsens co-infections with  human immunodeficiency virus (HIV).  This  drug stimulates the replication of several strains of HIV-1 in monocyte-derived DCs, and also renders such cells susceptible to HIV-2 infection.   We do not know how this  drug will also increase the efficacy of the reverse transcription process in COVID-19 [1].

Eflornithine is a difluoromethylated ornithine (DFMO) compound with antineoplastic activity. Eflornithine irreversibly inhibits ornithine decarboxylase, an enzyme required for polyamine biosynthesis, thereby inhibiting the formation and proliferation of tumor cells.  DFMO has been shown to slow the replication of certain RNA viruses in a cell culture model [2].

Copper Chelators

• Neocuproine is a chelator that has been used to treat copper overload in cultured cells.  However the neocuproine MSDS (material safety data sheet) presents neocuproine as a highly toxic compound in the whole human.  Neocuproine is toxic when inhaled or comes in contact with the skin.  Its target organs are central nervous system, kidneys, liver, and optic nerve.
• Bathocuproine is classified as an irritant by PubChem.  While neocuproine-Cu+ was shown to inhibit the HIV-1 protease needed to cleave the viral poly-protein into individual proteins.[3].  However Neo-Cu⁺  failed to inhibit HIV-1 poly-protein processing in a T helper cell line.  The authors proposed other methods of inhibition of viral replication.  While Coivd-19 also has a viral protease needed to cleave the poly-protein, we have no evidence that  inhibits the 3CL protease, or if it is possible to  achieve inhibitory concentrations by spraying this material into the lungs.  If nothing else, direct spraying into the lungs could limit the sites of side effects.

Forbes Recommended Covid-19 treatments

In April of this year hydroxychloroquine and azithromycin  were touted by Forbes Magazine as a possible Covid-19 combination treatment.

Hydroxychloroquine is an anti-malarial that may also be an irritant at the wrong dosing and route of exposure.

Azythithromax is an antibiotic that inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome.  PubChem reports rare cases of acute liver injury.  

A French study published at the end of March followed the  virologic and clinical outcomes of 11 consecutive patients hospitalized who received hydroxychloroquine (600 mg/d for 10 days) and azithromycin (500 mg day 1 and 250 mg days 2 to 5) using  previously dosing [4].  No benefit of this combination treatment was observed.  One patient had to discontinue treatment due to cardiac function abnormalities.  a subsequent study of 200 Covid-19 patients on the combination therapy or dihyrochloroquine alone revealed a greater incidence in long QT abnormalities.   And just two days of this writing, ” These medications can pathologically prolong the QT interval and lead to malignant ventricular arrhythmias such that organized guidance on QT evaluation and management strategies are important to reduce morbidity associated with the potential large-scale use.” [6]

The ACE2 inhibitor and cannabidiol

ACCE2 counter blances ACE1 in terms of maintaining blood pressure [7].  ACE2 expression may be influeced by ACE1 inhibitors [7].  In addition to the lungs, ACE2 is expressed in the brain, gut, and kidney.  The loss of ACE2 in the brain stem may facilitate an increase in sympathetic drive, alterations in the baroreflex, and exacerbation of hypertension.

And finally, there is some suggestion that CBD might make Covid-19 infections worse [8].

References

1. Barat C, Pepin J, Tremblay MJ. (2011) HIV-1 replication in monocyte-derived dendritic cells is stimulated by melarsoprol, one of the main drugs against human African trypanosomiasis. J Mol Biol. 2011 Jul 29;410(5):1052-64
2. Mounce BC, Cesaro T, Moratorio G, Hooikaas PJ, Yakovleva A, Werneke SW, Smith EC, Poirier EZ, Simon-Loriere E, Prot M, Tamietti C, Vitry S, Volle R, Khou C, Frenkiel MP, Sakuntabhai A, Delpeyroux F, Pardigon N, Flamand M, Barba-Spaeth G, Lafon M, Denison MR, Albert ML, Vignuzzi M. (2016)Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. J Virol. 2016 Oct 14;90(21):9683-9692. [PMC free article]
3. Davis DA, Branca AA, Pallenberg AJ, Marschner TM, Patt LM, Chatlynne LG, Humphrey RW, Yarchoan R, Levine RL. (1995)Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+. Arch Biochem Biophys. 322(1):127-34.
4. Molina JM, Delaugerre C, Le Goff J, Mela-Lima B, Ponscarme D, Goldwirt L, de Castro N.(2020)No evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe COVID-19 infection. Med Mal Infect. 2020 Jun;50(4):384. [PMC free article]
5. Saleh M, Gabriels J, Chang D, Kim BS, Mansoor A, Mahmood E, Makker P, Ismail H, Goldner B, Willner J, Beldner S, Mitra R, John R, Chinitz J, Skipitaris N, Mountantonakis S, Epstein LM.(2020 Apr 29) The Effect of Chloroquine, Hydroxychloroquine and Azithromycin on the Corrected QT Interval in Patients with SARS-CoV-2 Infection. Circ Arrhythm Electrophysiol.
6. Asensio E, Acunzo R, Uribe W, Saad EB, Sáenz LC.(2020) Recommendations for the measurement of the QT interval during the use of drugs for COVID-19 infection treatment. Updatable in accordance with the availability of new evidence. J Interv Card Electrophysiol. 2020 May 16
7. South AM, Diz DI, Chappell MC. (2020) COVID-19, ACE2, and the cardiovascular consequences. Am J Physiol Heart Circ Physiol. 2020 May 1;318(5):H1084-H1090. doi: 10.1152/ajpheart.00217.2020. Epub 2020 Mar 31.[PMC free article]  [author pod cast]
8. Brown JD. (2020) Cannabidiol as prophylaxis for SARS-CoV-2 and COVID-19? Unfounded claims versus potential risks of medications during the pandemic.Res Social Adm Pharm. 2020 Mar 31. pii: S1551-7411(20)30300-4. [Cross Ref]

2023 update: This is another password protected post from May of 2020. We were actually forward thinking that maybe Covid-19 could be made worse by mycoplasma coinfections. A PubMed search on Mollicute and Coivd-19 reveals as much. Some day we should look at Cu(I)NA₂ as an antibacterial for these cell wall lacking bacteria. In some ways this post to our founder who had this hunch.

Corona Viruses, Kawasaki’s Disease, and Children

Tang (2004) performed a PCR survey of pediatric cases of Kawasaki syndrome in Taiwan.   RNA and DNA was extracted from  throat swabs, and nasopharyngeal swabs.  Nucleic acid sequences to identify the pathogens were amplified using PCR primers  for enterovirus, adenovirus, influenza B, rhinovirus, metapneumovirus, and coronavirus.  These authors found that children with Kawasaki’s syndrome were more likely to to have infections with common respiratory viruses [1].

COVID-19 and Mollicutes

Mollicutes is the new term for bacteria, formerly called mycoplasmas, that lack cell walls.  Garth Nicolson, PhD and Gonzalo Ferreira de Mattos, MD, PhD and published a recent hypothesis paper proposing that COVID-19 deaths are due to coinfection with Mycoplasma pnuemoniae and related species of pathogenic bacteria [2].

Pneumonia is and inflammation of the aveoli of the lung that is generally caused by viral or bacterial infections.  Fungi and parasites may also cause pneumonia.

CDC page on Mycoplasma pneumoniae is a good resource.   Because these bacteria lack cell walls, therefore, β-lactam antibiotics, of which Penicillin is one, are of little use.  Macrolides are commonly prescribed to children and adults with Mycoplasma infections.  The CDC reports resistance as high as 90% in Asia with emerging cases of resistance in the United States and Europe.  The CDC makes diagnosing Mollicute infections sound difficult and time consuming.

As a look back to 2016, Mycoplasma penumoniae was found in close to 14%  of Kawasaki cases in the Children’s Hospital of Soochow University, China.  These authors did not look for viral pathogens that were on the scene back in 2016. [4]

The CDC’s list of Mycoplasma pneumoniae Treatments

These antibiotics came from the CDC’s website with additional information of Wikipedia websites.

• Macrolides (e.g., azithromycin): Children and adults  Macrolides are generally considered the treatment of choice. However, clinicians should practice prudent use of macrolide drugs due to the emergence of macrolide-resistant strains of M. pneumoniae.   Macrolides inhibit the translation of mRNA into proteins in bacteria.
• Fluoroquinolones: Adults  Fluoroquinolones inhibit DNA replication in bacteria.  Unfortunately they have neurological side effects in humans.
• Tetracyclines (e.g., doxycycline): Older children and adults  Tetracyclines also inhibit the translation of mRNA into proteins in bacteria.

Clinicians should not prescribe fluoroquinolones and tetracyclines for young children under normal circumstances.

Concluding remarks

Mollicute and Covid-19 co-infections seem to be an emerging problem.  Cases of Kawasaki Disease linked to mollicute and corona virus family infections raises some interesting questions.  Could the right copper boost to the immune system prevent the use of antibiotics when bacterial co-infections are simply prolonging recovery?

References

1. Raha S, Mallick R, Basak S, Duttaroy AK.(2020) Is copper beneficial for COVID-19 patients? Med Hypotheses. 2020 May 5;142:109814. [PMC free article]
2. Chang LY, Lu CY, Shao PL, Lee PI, Lin MT, Fan TY, Cheng AL, Lee WL, Hu JJ, Yeh SJ, Chang CC, Chiang BL, Wu MH, Huang LM. (2014)Viral infections associated with Kawasaki disease. J Formos Med Assoc. 2014 Mar;113(3):148-54. [PMC free article]
3. Nicolson GL, Ferreira de Mattos G (2020) COVID-19 Coronavirus: Is Infection Along with Mycoplasma or other Bacteria Linked to Progression to a Lethal Outcome? International Journal of Clinical Medicine 11: [Cross Ref]
4. Tang Y, Yan W, Sun L, Huang J, Qian W, Hou M, Lv H.(2016) Kawasaki disease associated with Mycoplasma pneumoniae. Ital J Pediatr. 42(1):83  [PMC free article]