Back to the Basics of SARS-CoV-2 Biochemistry: Microvascular Occlusive Glycan Bindings Govern Its Morbidities and Inform Therapeutic Responses.

Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three-SARS, SARS-CoV-2 and MERS-are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them.

Increased von Willebrand and Factor VIII plasma levels in gynecologic patients with Post-Acute-COVID-Sequela (PASC)/Long COVID.

Up to 30 % of COVID-infected patients may develop post-acute sequelae of COVID-19 (PASC), also known as Long COVID (LC), a syndrome characterized by a variety of debilitating symptoms lasting for more than 3 months after the acute infection. While the pathophysiological mechanisms behind PASC/LC are not completely understood, growing evidence suggests that an important component of this syndrome may be related to persistent microvascular inflammation causing clumping/clotting of red blood cells and platelets and thrombotic complications. We retrospectively evaluated the plasma levels of von Willebrand factor (VWF), Factor VIII and D-dimer in 10 gynecologic patients (60 % with an endometrial or ovarian cancer diagnosis) affected by PASC/LC vs 5 control patients (60 % harboring endometrial or ovarian tumors). We found elevated VWF and Factor VIII levels in all 10 PASC/LC patients (means of 254 % and 229 %, respectively) vs none of the 5 randomly selected cancer control patients (means of 108 % and 95 %, respectively), p = 0.0046 and p < 0.0001, respectively. In contrast, no significant difference was noted in the levels of D-dimer in PASC/LC. Importantly, abnormally elevated VWF and Factor VIII levels were found to persist for at least 2 years in patients with Long COVID symptoms. VWF and Factor VIII but not D-dimer levels are significantly elevated in the plasma of PASC/LC cancer patients. Abnormally and persistently elevated VWF and Factor VIII levels may represent the results of persistent microvascular damage (i.e., spike-induced endotheliosis) and may be biomarkers of persistent inflammation in gynecologic patients with PASC/LC.

Sialylated Glycan Bindings from SARS-CoV-2 Spike Protein to Blood and Endothelial Cells Govern the Severe Morbidities of COVID-19.

Consistent with well-established biochemical properties of coronaviruses, sialylated glycan attachments between SARS-CoV-2 spike protein (SP) and host cells are key to the virus's pathology. SARS-CoV-2 SP attaches to and aggregates red blood cells (RBCs), as shown in many pre-clinical and clinical studies, causing pulmonary and extrapulmonary microthrombi and hypoxia in severe COVID-19 patients. SARS-CoV-2 SP attachments to the heavily sialylated surfaces of platelets (which, like RBCs, have no ACE2) and endothelial cells (having minimal ACE2) compound this vascular damage. Notably, experimentally induced RBC aggregation in vivo causes the same key morbidities as for severe COVID-19, including microvascular occlusion, blood clots, hypoxia and myocarditis. Key risk factors for COVID-19 morbidity, including older age, diabetes and obesity, are all characterized by markedly increased propensity to RBC clumping. For mammalian species, the degree of clinical susceptibility to COVID-19 correlates to RBC aggregability with p = 0.033. Notably, of the five human betacoronaviruses, the two common cold strains express an enzyme that releases glycan attachments, while the deadly SARS, SARS-CoV-2 and MERS do not, although viral loads for COVID-19 and the two common cold infections are similar. These biochemical insights also explain the previously puzzling clinical efficacy of certain generics against COVID-19 and may support the development of future therapeutic strategies for COVID-19 and long COVID patients.

COVID-19 Excess Deaths in Peru's 25 States in 2020: Nationwide Trends, Confounding Factors, and Correlations With the Extent of Ivermectin Treatment by State.

Introduction In 2020, nations hastened to contain an emerging COVID-19 pandemic by deploying diverse public health approaches, but conclusive appraisals of the efficacy of these approaches are elusive in most cases. One of the medicines deployed, ivermectin (IVM), a macrocyclic lactone having biochemical activity against SARS-CoV-2 through competitive binding to its spike protein, has yielded mixed results in randomized clinical trials (RCTs) for COVID-19 treatments. In Peru, an opportunity to track the efficacy of IVM with a close consideration of confounding factors was provided through data for excess deaths as correlated with IVM use in 2020, under semi-autonomous policies in its 25 states. Methods To evaluate possible IVM treatment effects, excess deaths as determined from Peruvian national health data were analyzed by state for ages ≥60 in Peru's 25 states. These data were compared with monthly summary data for excess deaths in Peru for the period 2020-2021 as published by the WHO in 2022. To identify potential confounding factors, Google mobility data, population densities, SARS-CoV-2 genetic variations, and seropositivity rates were also examined. Results Reductions in excess deaths over a period of 30 days after peak deaths averaged 74% in the 10 states with the most intensive IVM use. As determined across all 25 states, these reductions in excess deaths correlated closely with the extent of IVM use (p<0.002). During four months of IVM use in 2020, before a new president of Peru restricted its use, there was a 14-fold reduction in nationwide excess deaths and then a 13-fold increase in the two months following the restriction of IVM use. Notably, these trends in nationwide excess deaths align with WHO summary data for the same period in Peru. Conclusions The natural experiment that was put into motion with the authorization of IVM use for COVID-19 in Peru in May 2020, as analyzed using data on excess deaths by locality and by state from Peruvian national health sources, resulted in strong evidence for the drug's effectiveness. Several potential confounding factors, including effects of a social isolation mandate imposed in May 2020, variations in the genetic makeup of the SARS-CoV-2 virus, and differences in seropositivity rates and population densities across the 25 states, were considered but did not appear to have significantly influenced these outcomes.

Computational Prediction of the Interaction of Ivermectin with Fibrinogen.

Hypercoagulability and formation of extensive and difficult-to-lyse microclots are a hallmark of both acute COVID-19 and long COVID. Fibrinogen, when converted to fibrin, is responsible for clot formation, but abnormal structural and mechanical clot properties can lead to pathologic thrombosis. Recent experimental evidence suggests that the spike protein (SP) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may directly bind to the blood coagulation factor fibrinogen and induce structurally abnormal blood clots with heightened proinflammatory activity. Accordingly, in this study, we used molecular docking and molecular dynamics simulations to explore the potential activity of the antiparasitic drug ivermectin (IVM) to prevent the binding of the SARS-CoV-2 SP to fibrinogen and reduce the occurrence of microclots. Our computational results indicate that IVM may bind with high affinity to multiple sites on the fibrinogen peptide, with binding more likely in the central, E region, and in the coiled-coil region, as opposed to the globular D region. Taken together, our in silico results suggest that IVM may interfere with SP-fibrinogen binding and, potentially, decrease the formation of fibrin clots resistant to degradation. Additional in vitro studies are warranted to validate whether IVM binding to fibrinogen is sufficiently stable to prevent interaction with the SP, and potentially reduce its thrombo-inflammatory effect in vivo.

When Characteristics of Clinical Trials Require Per-Protocol as Well as Intention-to-Treat Outcomes to Draw Reliable Conclusions: Three Examples.

Under exceptional circumstances, including high rates of protocol non-compliance, per-protocol (PP) analysis can better indicate the real-world benefits of a medical intervention than intention-to-treat (ITT) analysis. Exemplifying this, the first randomized clinical trial (RCT) considered found that colonoscopy screenings were marginally beneficial, based upon ITT analysis, with only 42% of the intervention group actually undergoing the procedure. However, the study authors themselves concluded that the medical efficacy of that screening was a 50% reduction in colorectal cancer deaths among that 42% PP group. The second RCT found a ten-fold reduction in mortality for a COVID-19 treatment drug vs. placebo by PP analysis, but only a minor benefit by ITT analysis. The third RCT, conducted as an arm of the same platform trial as the second RCT, tested another COVID-19 treatment drug and reported no significant benefit by ITT analysis. Inconsistencies and irregularities in the reporting of protocol compliance for this study required consideration of PP outcomes for deaths and hospitalizations, yet the study coauthors refused to disclose them, instead directing inquiring scientists to a data repository which never held the study's data. These three RCTs illustrate conditions under which PP outcomes may differ significantly from ITT outcomes and the need for data transparency when these reported or indicated discrepancies arise.

SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects.

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen.

A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at Its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody.

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.

Cytotoxicity of unsaturated fatty acids in fresh human tumor explants: concentration thresholds and implications for clinical efficacy.

BACKGROUND: Unsaturated fatty acids (UFAs) exhibit in vitro cytotoxicity against many malignant cell lines and yield decreased cancer incidence and reduced tumor growth in animal models. But clinical and animal studies to date have achieved response using only localized delivery methods such as intratumoral infusion. To explore possibilities for enhanced clinical efficacy, fresh surgical explants of tumors from 22 patients with five malignancies were exposed to gamma-linolenic acid (GLA) and alpha-linolenic acid (ALA) and analyzed with an in vitro chemosensitivity testing system, the Fluorescent Cytoprint Assay (FCA). A total of 282 micro-organ cultures derived from these malignant tumors were exposed to GLA and ALA at different concentrations. RESULTS: GLA and ALA exhibited greater than 90% cytotoxicity at a sharp concentration threshold between 500 microM and 1 mM against all but two malignant micro-organ cultures tested in 5-10% serum. In tests using 30-40% serum, GLA and ALA killed tumor at concentrations of 2 mM and above. CONCLUSIONS: The concentration threshold of 500 microM to 2 mM exhibited for antitumor activity by GLA and ALA is much higher than that observed in most previously reported cell culture studies but consistent with physiological concentrations found to kill tumor clinically and in animals. A mechanism of antitumor activity by unsaturated fatty acids through selective destabilization of the malignant plasma membrane is considered. An oral regimen is proposed for phase I clinical testing that could push the area under the curve for serum concentration of unbound unsaturated fatty acids over time to much higher levels than previously achieved for systemic administration and into the range that could yield antitumor response.