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mahesh d
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  • Load the DNA samples dissolved in 6x alkaline gel-loading buffer into the wells of the gel.
  • Alkaline gels draw more current than neutral gels at comparable voltages and heat up during the run. Alkaline agarose electrophoresis should therefore be carried out at <3.5 V/cm.
  • Do not add ethidium bromide because the dye will not bind to DNA at high pH. The addition of NaOH to a hot agarose solution causes hydrolysis of the polysaccharide. For this reason, the agarose is first melted in H2O and then made alkaline by the addition of NaOH just before the gel is poured.
  • Cool the clear solution to 55'C. Add 0.1 volume of 10x alkaline agarose gel electrophoresis buffer, and immediately pour the gel.After the gel is completely set, mount it in the electrophoresis tank and add freshly made 1x alkaline electrophoresis buffer until the gel is just covered.
  • Alkaline agarose gels are run at a pH that is sufficiently high  to denature double-stranded DNA. The denatured DNA is maintained  in a single-stranded state and migrates through the alkaline  gel as a function of its size.
  • yes-  EtBr does intercalate very effectively in dsDNA - now if you denature the same DNA to ssDNA - the bases dont necessarily form a perfect duplex. however to protect its hydrophobic bases from the water around, it does attain arbitrary sec structures by non specific pairing. Those non specific hydrophobic cores are enough to trap EtBr and increase its fluorescent yield which one will see as fluroescence. However, one must note - that the EtBr binding is going to be quantitatively SIGNIFICANTLY lesser to ssDNA of the same size and concentration as that of a corresponding dsDNA. Having said all this - I must add that EtBr fluor/binding to ssDNA is not quantitative and varies depending on gel conditions. The fluctuation is much lesser for regular dsDNA. 
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  • The mRNA COVID-19 vaccines by Pfizer and Moderna have undergone safety testing in human clinical trials
  • It is not possible for mRNA to move into the nucleus of a cell as it lacks the signals that would allow it to enter this compartment. This means that RNA cannot integrate into the DNA of the vaccinated cell
  • ribonucleases (RNases)Trusted Source degrade the mRNA.
  • Encapsulating mRNA in lipid nanoparticles
  • Delivering mRNA successfully to cells inside our bodies and ensuring that enzymes within our cells do not degrade it are key challenges in vaccine development. Chemical modifications
  • Both mRNA COVID-19 vaccines that Pfizer/BioNTech and Moderna have developed cannot cause COVID-19. They do not carry the full information for our cells to make the SARS-CoV-2 virus, and therefore, cannot cause an infection.
  • Importantly, mRNA vaccines only carry the information to make a small part of a pathogen. From this information, it is not possible for our cells to make the whole pathogen.
  • Recombinant vaccine technology employs yeast or bacterial cells to made many copies of a particular viral or bacterial protein or sometimes a small part of the protein. mRNA vaccines bypass this step. They are chemically synthesized without the need for cells or pathogens, making the production process simpler. mRNA vaccines carry the information that allows our own cells to make the pathogen’s proteins or protein fragments themselves
  • An mRNA vaccine delivers the instructions for making a bacterial or viral protein to our cells. Our immune system then responds to these proteins and develops the tools to react to future infections with the pathogen. mRNA vaccine technology is not new, but there were no mRNA vaccines that had approval for use in humans until recently.
  • Most vaccines contain an infectious pathogen or a part of it, but mRNA vaccines deliver the genetic instructions for our cells to make viral or bacterial proteins themselves. Our immune system responds to these and builds up immunity
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  • With all viral vectors, one issue to consider is preexisting immunity. If a person encountered the virus that serves as the vector in the past, they may have antibodies to the virus. This means that their body will try to fight and destroy the viral vector, potentially making a vaccine less effective.The Oxford University research team behind the Oxford-AstraZeneca COVID-19 vaccine previously reportedTrusted Source that levels of preexisting antibodies to the ChAdOx1 viral vector were low when they assessed this in samples from adults from the United Kingdom and Gambia.
  • Other COVID-19 vaccines that use viral vectors include the Russian Sputnik V vaccine and the Janssen single-dose vaccine candidate.
  • Then our cells present the spike protein, as well as small parts of it, on the cell surface, prompting our immune system to make antibodies and mount T cell responses.
  • Our cells then transcribe this gene into messenger RNA, or mRNA, which in turn prompts our cellular machine to make the spike protein in the main body, or the cytoplasm, of the cell.
  • chimpanzee adenoviral vector
  • Originally, researchers worked with modified adenoviruses for the purpose of gene therapy. However, because they are able to stimulate our immune system, adenoviral vectors make good candidates for vaccine development.
  • The Oxford-AstraZeneca COVID-19 vaccine uses a chimpanzee common cold viral vector known as ChAdOx1, which delivers the code that allows our cells to make the SARS-CoV-2 spike protein.
  • However, the viral vector itself plays an additional role by boosting our immune response
  • Our cells then make the viral or bacterial protein that the vector has delivered and present it to our immune system
  • Viral vector vaccines work differently. They make use of a harmless virus to deliver a piece of genetic code from a pathogen to our cells to mimic an infection. The harmless virus acts as a delivery system, or vector, for the genetic sequence
  • When we have a bacterial or viral infection, our immune system reacts to molecules from the pathogen. If it is our first encounter with the invader, a finely tuned cascade of processes come together to fight the pathogen and build up immunity for future encounters
  • viral vector vaccines work?
  • viral vector vaccines use a harmless virus to deliver a piece of genetic code to our cells, allowing them to make a pathogen’s protein. This trains our immune system to react to future infections.
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  • Overall, according to Bai et al (17), compared to non-COVID-19 viral pneumonia, parenchymal opacities in COVID-19 pneumonia were more likely to be peripheral (80% vs 57%), and have GGO (91% vs 68%), fine reticular opacity (56% vs 22%) and vascular thickening (11% vs 1%). COVID-19 patients were less likely to have central and peripheral distribution (14% vs 35%), air bronchograms (14% vs 23%), pleural thickening (15% vs 33%), pleural effusion (4% vs 39%) and lymphadenopathy (2.7% vs 10.2%) (17
  • Li et al (20) explored differences in CT features of COVID-19 versus other Coronaviridae, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). They report that GGO, consolidation, septal thickening, and air bronchogram sign were similar in COVID-19, SARS, and MERS, whereas reversed halo sign and pulmonary nodules associated with COVID-19 have not been previously described with SARS and MERS. Lung abnormalities in SARS are more commonly reported to be unifocal (40).
  • The viral infections most often described to have features that resemble COVID-19 include influenza, cytomegalovirus, and other coronaviruses (38–41). With regard to the differentiation of COVID-19 from influenza, Liu et al (38) found that, although peripheral GGOs and consolidation are seen in both these entities, round opacities and septal thickening are more common in COVID-19. Conversely, nodules, tree-in-bud opacities, and pleural effusion are more common in influenza (38).
  • Viruses are the most common causes of respiratory tract infections and are seen more commonly in children, the elderly, and the immunocompromised (33,34). The most common pathogen causing viral pneumonia in both immunocompetent and immunocompromised patients is influenza virus (33). The clinical signs and symptoms of viral pneumonia are often diverse and depend on host immune status (34). The spectrum of CT findings encountered in various pulmonary viral diseases encompasses four main categories: (a) GGO and consolidation; (b) nodules, micronodules, and tree-in-bud opacities; (c) interlobular septal thickening; and (d) bronchial and/or bronchiolar wall thickening (35) (Fig 3). Lymphadenopathy and pleural effusions may also be present (36). Some of the viral pneumonias can manifest as substantial GGO and include cytomegalovirus, adenovirus, herpes simplex virus, varicella zoster, measles, human meta-pneumovirus, and influenza (33,37). Percentage area of lung involvement with GGOs with different viruses has been extensively described. GGOs can be seen in 50%–75% of patients with adenovirus, in more than 75% of patients with cytomegalovirus and herpes simplex virus, and in 10%–25% of patients with human meta-pneumovirus and measles (33).
  • In contrast to COVID-19, bacterial pneumonia characteristically produces focal segmental or lobar pulmonary opacities without lower lung predominance. Complications or associated findings such as cavitation, lung abscess, lymphadenopathy, parapneumonic effusions and empyema, when present, are useful imaging differentiating features, as they are not seen in COVID-19 unless the patients are superinfected with bacterial pneumonia
  • cavitation
  • Asymptomatic carriers of COVID-19 may comprise 17.9%–33.3% of infected cases (28,29). Such patients must be directed toward RT-PCR testing.
  • Chest CT in COVID-19 pneumonia demonstrates bilateral, peripheral, and basal predominant ground-glass opacities (GGOs) and/or consolidation in nearly 85% of patients with superimposed irregular lines and interfaces; the imaging findings peak 9–13 days after infection (7,8) (Fig 1). Subsequently, a mixed pattern evolves with crazy paving, architectural distortion, and perilobular abnormalities superimposed on GGOs with slow resolution (7
  • Chest CT findings in coronavirus disease 2019 (COVID-19) pneumonia are variable but can be bilateral, lower lobe, and extend to the pleural surfaces; these features can be helpful in distinguishing COVID-19 pneumonia from other causes of lung abnormality.
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  • and homeostatic capacity
  • appears to function in both an inflammatory
  • PGD2 is the predominant prostanoid produced by activated mast cells, which initiate IgE-mediated Type I acute allergic responses (104, 111). It is well established that the presence of an allergen triggers the production of PGD2 in sensitized individual
  • PGI2 is a potent vasodilator, and an inhibitor of platelet aggregation, leukocyte adhesion, and VSMC proliferation (75). PGI2 is also anti-mitogenic and inhibits DNA synthesis in VSMC (83). These actions of PGI2 are mediated through specific IP receptors (Table). This receptor is expressed in kidney, liver, lung, platelets, heart, and aorta
  • In vivo studies in mice and humans showed that COX-2 was the dominant source of PGI2 (81).
  • Prostaglandin I2 and inflammationPGI2 is one of the most important prostanoids that regulates cardiovascular homeostasis. Vascular cells, including endothelial cells, vascular smooth muscle cells (VSMCs) and endothelial progenitor cells (EPCs), are the major source of PGI2 (76).
  • Prostaglandin E2 and inflammationPGE2 is one of the most abundant PGs produced in the body, is most widely characterized in animal species, and exhibits versatile biological activities. Under physiological conditions, PGE2 is an important mediator of many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility. Dysregulated PGE2 synthesis or degradation has been associated with a wide range of pathological conditions (36). In inflammation, PGE2 is of particular interest because it is involved in all processes leading to the classic signs of inflammation: redness, swelling and pain (37). Redness and edema result from increased blood flow into the inflamed tissue through PGE2-mediated augmentation of arterial dilatation and increased microvascular permeability (37). Pain results from the action of PGE2 on peripheral sensory neurons and on central sites within the spinal cord and the brain (37).
  • Prostaglandin production (Figure 1) depends on the activity of prostaglandin G/H synthases, colloquially known as COXs, bifunctional enzymes that contain both cyclooxygenase and peroxidase activity and which exist as distinct isoforms referred to as COX-1 and COX-2 (2). COX-1, expressed constitutively in most cells, is the dominant source of prostanoids that subserve housekeeping functions, such as gastric epithelial cytoprotection and homeostasis (3). COX-2, induced by inflammatory stimuli, hormones and growth factors, is the more important source of prostanoid formation in inflammation and in proliferative diseases, such as cancer (3). However, both enzymes contribute to the generation of autoregulatory and homeostatic prostanoids, and both can contribute to prostanoid release during inflammation.
  • The two cyclooxygenase isoforms, COX-1 and COX-2, are targets of nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs are competitive active site inhibitors of both COXs.
  • Prostaglandins and thromboxane A2 (TXA2), collectively termed prostanoids
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  • Antithrombin is a serine protease inhibitor (serpin) that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa.
  • All mammals have an extremely closely related blood coagulation process, using a combined cellular and serine protease process.[citation needed] In fact, it is possible for any mammalian coagulation factor to "cleave" its equivalent target in any other mammal.
  • The only non-mammalian animal known to use serine proteases for blood coagulation is the horseshoe crab
  • Coagulation factors
  • Anticoagulants[edit] Main articles: Antiplatelet drug and Anticoagulant Anticoagulants and anti-platelet agents are amongst the most commonly used medications. Anti-platelet agents include aspirin, dipyridamole, ticlopidine, clopidogrel, ticagrelor and prasugrel; the parenteral glycoprotein IIb/IIIa inhibitors are used during angioplasty. Of the anticoagulants, warfarin (and related coumarins) and heparin are the most commonly used. Warfarin affects the vitamin K-dependent clotting factors (II, VII, IX, X) and protein C and protein S, whereas heparin and related compounds increase the action of antithrombin on thrombin and factor Xa. A newer class of drugs, the direct thrombin inhibitors, is under development; some members are already in clinical use (such as lepirudin). Also in clinical use are other small molecular compounds that interfere directly with the enzymatic action of particular coagulation factors (the directly acting oral anticoagulants: dabigatran, rivaroxaban, apixaban, and edoxaban).
  • Thrombosis is the pathological development of blood clots.
  • In acute or chronic liver failure, there is insufficient production of coagulation factors, possibly increasing risk of bleeding during surgery.
  • mediate the activation of platelets and formation of primary hemostasis
  • Von Willebrand disease
  • The best-known coagulation factor disorders are the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease") and hemophilia C (factor XI deficiency, mild bleeding tendency).
  • Many acute-phase proteins of inflammation are involved in the coagulation system. In addition, pathogenic bacteria may secrete agents that alter the coagulation system, e.g. coagulase and streptokinase.
  • lso, some products of the coagulation system can contribute to the innate immune system by their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells.
  • In addition, some of the products of the coagulation system are directly antimicrobial. For example, beta-lysine, an amino acid produced by platelets during coagulation, can cause lysis of many Gram-positive bacteria by acting as a cationic detergent.
  • Coagulation can physically trap invading microbes in blood clots.
  • Eventually, blood clots are reorganised and resorbed by a process termed fibrinolysis. The main enzyme responsible for this process (plasmin)
  • Prostacyclin (PGI2) is released by endothelium and activates platelet Gs protein-linked receptors. This, in turn, activates adenylyl cyclase, which synthesizes cAMP. cAMP inhibits platelet activation by decreasing cytosolic levels of calcium and, by doing so, inhibits the release of granules that would lead to activation of additional platelets and the coagulation cascade.[12]
  • Plasmin is generated by proteolytic cleavage of plasminogen, a plasma protein synthesized in the liver. This cleavage is catalyzed by tissue plasminogen activator (t-PA), which is synthesized and secreted by endothelium. Plasmin proteolytically cleaves fibrin into fibrin degradation products that inhibit excessive fibrin formation.
  • The activated platelets change shape from spherical to stellate, and the fibrinogen cross-links with glycoprotein IIb/IIIa aid in aggregation of adjacent platelets (completing primary hemostasis).[6]
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  • In COVID-19 patients, secondary HLH and cytokine storm may be responsible for unexplained progressive fever, cytopenia, ARDS, neurological and renal impairment. Differentiation between the primary and secondary forms of HLH is utterly important, since primary form of HLH requires complicated treatments such as hematopoietic stem cell transplantation
  • The selection is restricted to the past 5 years and limited numbers of earlier key references were manually selected.
  • MED-LINE/Pubmed was searched from inception to April 2020, and the following terms were used for data searching: "hemophagocytic syndrome" OR "macrophage activation syndrome" OR "hemophagocytic lymphohistiocytosis", OR "cytokine storm". Finally, AND "COVID-19" was included in this algorithm.
  • This paper aims to review the pathogenesis and the clinical picture of HLH, and its severe complication, the cytokine storm, with a special emphasis on the developed classification criteria sets for rheumatologists, since COVID-19 infection has clinical symptoms resembling those of the common rheumatologic conditions and possibly triggers HLH.
  • Hemophagocytic syndrome (HPS) or hemophagocytic lymphohistiocytosis (HLH) is an acute and rapidly progressive systemic inflammatory disorder characterized by cytopenia, excessive cytokine production, and hyperferritinemia. Common clinical manifestations of HLH are acute unremitting fever, lymphadenopathy, hepatosplenomegaly, and multiorgan failure. Due to a massive cytokine release, this clinical condition is considered as a
  • cytokine storm syndrome
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  • Given their interaction with immune cells, endothelium, and clotting factors, and the widespread use of antiplatelet drugs in the general population, platelets seem an appealing therapeutic target in sepsis. Data from studies of both animal models and septic patients have shown the contribution of platelets to multi-organ dysfunction
  • Antiplatelets and Prevention of Organ Damage during Sepsis
  • ASA inhibits platelet function through the blocking of COX-1. Clopidogrel is a platelet membrane P2Y12 receptor inhibitor, thereby preventing ADP (adenosine diphosphate) activation. Antiplatelet drugs have an important anti-inflammatory effect, and can reduce C-reactive protein, P-selectin, and leukocyte-platelet aggregates [76,77], and have therefore been proposed as possible targets for sepsis prevention and treatment.
  • although nitric oxide modulation and leukocyte recruitment have also been suggested in the pathogenesis
  • sepsis induced cardiac impairment resolves within 7–10 days.
  • Blocking P-selectin protects mice from AKI by attenuating neutrophil recruitment into the kidney
  • MPs are an interesting pathogenetic mechanism, and a correlation between MPs and blood urea nitrogen in AKI has been indicated in septic patients
  • NET inhibition seems to be the most appealing approach, in particular inhibition of the NET associated protein histone H4, which has been shown to protect from DIC in an animal model of sepsis
  • Also, intravenous DNAse has shown to successfully breakdown NETs and reduce organ damage
  • . NETs mainly play a role in very small vessels, including lung capillary and hepatic sinusoids.
  • a complex web-like structure of DNA with proteolytic activity built by neutrophils, with the ability to trap microorganisms and facilitate their clearance
  • Platelets are also involved in the formation of
  • Neutrophil extracellular traps (NETs)
  • In particular, platelet-neutrophil aggregates are platforms for thrombi generation and that is the trigger for NET release
  • About 80% of all septic patients have some degree of coagulopathy. DIC (disseminated intravascular coagulation) is a condition involving uncontrolled systemic activation of the clotting cascade leading to clotting factor consumption and microvascular thrombosis. Complications include thrombotic and hemorrhagic events.
  • platelet depletion has been shown to correlate with reduced recruitment of neutrophils in lung interstitium [63] and increased platelet-derived thromboxane-A2 and P-selectin correlated with increased neutrophil activation, in a mouse model of ARDS [28]. This second mechanism can be reversed, as shown by inhibition of P-selectin with antibodies and in knock out mice model of barotrauma
  • Leukocytes and platelet recruitment, intravascular coagulation, endothelial damage, loss of surfactant, oxidative stress are all mechanisms underlying severe lung damage in sepsis.
  • Post mortem biopsies of patients who died with ARDS have shown excess numbers of platelets and neutrophil deposition in pulmonary vessels [62].
  • lung protective ventilation to reduce lung trauma generated by high pressure in alveoli. Barotrauma is correlated with increased inflammation and worse prognosis
  • Acute respiratory distress syndrome (ARDS) is one the most severe complications of sepsis and is characterised by increased alveolar-capillary barrier permeability, pulmonary oedema, and severe hypoxemia
  • Interesting new evidence of the possible benefit of antioxidants and radical oxygen species scavengers (e.g., high doses of intravenous vitamin C in septic shock [58]) have recently been published and appear promising [59,60]. However, further studies on reversal of mitochondrial dysfunction in sepsis are needed.
  • These findings support the idea that organ damage occurs at a subcellular level driven by oxidative stress and impaired mitochondrial respiration. Mitochondrial dysfunction has also been shown in platelets during sepsis; alterations in platelet mitochondrial respiration correlate with the severity of disease
  • Skeletal muscle mitochondrial dysfunction has been shown in both animal models
  • Capillaries, arterioles, venules, and micro-lymphatics are all part of the microvascular network. During sepsis, even when organ perfusion is preserved, patchy areas of reduced oxygen delivery and extraction and functional shunting have been shown
  • Immunothrombosis contributes to microvascular dysfunction, which is a hallmark of organ damage in sepsis
  • known as immunothrombosis
  • Platelet-endothelial adhesion, platelet-leukocyte aggregates, and NETs all contribute to the formation of microthrombi in small vessels. The cells involved release cytokines and chemokines resulting in further cellular recruitment, which can become a pathological self-sustaining dysregulated process resulting in septic shock.
  • To date, none of these mediators have been successfully targeted in clinical practice. However, future benefits may result from further characterization of the molecular and cellular mechanisms involved in these processes and contribute to a theragnostic approach to treating sepsis and improving mortality.
  • it has been shown that inhibition of TPO prevents lung, liver, and gut damage in a cecal ligation and puncture (CLP) model of sepsis
  • its possible role as a biomarker and pathogenic mediator of sepsis,
  • Our group and others have shown significantly elevated levels of TPO in both murine and human sepsis
  • it promotes platelet production through megakaryocyte stimulation and is released by platelets themselves upon activation. TPO levels are also increased during inflammatory states
  • TPO is the growth hormone involved in thrombopoiesis
  • Elevated MP levels correlate with the severity of sepsis in clinical studie
  • microparticles (MPs). MPs are small vesicles released from the cell surface of platelets, which function as storage for coagulation factors and cytokines
  • Animal models of severe bacterial sepsis have found that intravenous treatment with DNase reduces organ damage and improves surviva
  • NET biomarkers (free DNA/myeloperoxidase complexes)
  • Activated platelets interact with other cells via two main mechanisms: (1) expression of receptors on cellular surface; and, (2) release of cytoplasmic granules that contain immunomodulatory proteins.
  • Reduced numbers of PLCs are associated with progression of MOF [18,19] and although causality is yet to be demonstrated, it may represent an indirect sign of platelet consumption in vessels.
  • Platelets play an important role in the guidance and activation of neutrophils, supporting leukocyte rolling, adhesion, and transmigration in peripheral vessels.
  • Leukocytes that interact with platelets express a higher number of receptors related to infection and inflammation and have a stronger bactericidal capacity.
  • In this review the following questions will be considered: What are the possible mechanisms of platelet dysfunction leading to multi-organ failure during sepsis? What evidence do we have for these mechanisms? Is platelet function a potential therapeutic target in sepsis?
  • Platelets are able to release cytokines, recruit leukocytes, interact with bacteria and the endothelium, and contribute to microthrombi formation [16]. These mechanisms are adaptive and protective in the context of a localized infection, but become dysregulated and “maladaptive” during sepsis, contributing to organ damage
  • Platelets are anucleated cells that play an established role in hemostasis and coagulation. However, hemorrhagic complications during sepsis are rare and rarely lead to death.
  • the new definition of sepsis, organ dysfunction and a dysregulated immune host response are the key factors that differentiate infection from sepsis
  • Septic shock is characterised by a dysregulated inflammatory response, which can impair the microcirculation and lead to organ injury. Being at the crossroads between the immune system, clotting cascade, and endothelial cells, platelets seem to be an appealing central mediator and possible therapeutic target in sepsis.
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  • owever, CD63, actin, tubulin and various other cytoplasmatic proteins are not present in NETs.
  • Analysis by immunofluorescence corroborated that NETs contain proteins from azurophilic granules (neutrophil elastase, cathepsin G and myeloperoxidase), specific granules (lactoferrin), tertiary granules (gelatinase), and the cytoplasm;
  • kill invading pathogens through two strategies: engulfment of microbes and secretion of anti-microbials
  • Neutrophils
  • Upon in vitro activation with the pharmacological agent phorbol myristate acetate (PMA), Interleukin 8 (IL-8) or lipopolysaccharide (LPS), neutrophils release granule proteins and chromatin to form an extracellular fibril matrix known as NET through an active process.[2]
  • In 2004, a novel third function was identified: formation of NETs. NETs allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells
  • Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens
  • NETs would be formed in tissues at a site of bacterial/yeast infection, NETs have also been shown to form within blood vessels during sepsis (specifically in the lung capillaries and liver sinusoids)
  • Intra-vascular NET formation is tightly controlled and is regulated by platelets, which sense severe infection via platelet TLR4 and then bind to and activate neutrophils to form NETs. Platelet-induced NET formation occurs very rapidly (in minutes) and may or may not result in death of the neutrophils
  • More recently, it has also been shown that not only bacteria but also pathogenic fungi such as Candida albicans induce neutrophils to form NETs that capture and kill C. albicans hyphal as well as yeast-form cells.[7] NETs have also been documented in association with Plasmodium falciparum infections in children
  • NETs provide for a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly independent of phagocytic uptake. In addition to their antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of the pathogens. Furthermore, delivering the granule proteins into NETs may keep potentially injurious proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of inflammation
  • NETs disarm pathogens with antimicrobial proteins such as neutrophil elastase, cathepsin G and histones that have a high affinity for DNA
  • however, there are key differences in stimuli, timing, and ultimate end result
  • NET activation and release, or NETosis, is a dynamic process that can come in two forms, suicidal and vital NETosis.
  • suicidal NETosis can take hours, even with high levels of PMA stimulation, while vital NETosis that can be completed in a matter of minutes.[10]
  • Vital NETosis
  • Evidence from laboratory experiments suggests that NETs are cleaned away by macrophages that phagocytose and degrade them.[14]
  • The formation of NETs is regulated by the lipoxygenase pathway – during certain forms of activation (including contact with bacteria) neutrophil 5-lipoxygenase forms 5-HETE-phospholipids that inhibit NET formation
  • NETs are capable of capturing HIV virions and destroying them.[20] There is an increase in NET production throughout the course of HIV/SIV, which is reduced by ART. In addition, NETs are able to capture and kill various immune cell groups such as CD4+ and CD8+ T cells, B cells, and monocytes. This effect is seen not only with neutrophils in the blood, but also in various tissues such as the gut, lung, liver, and blood vessels.
  • NETs have also been associated with the production of IgG antinuclear double stranded DNA antibodies in children infected with P. falciparum malaria.[8] NETs have also been found in cancer patients.[18] Preclinical research suggests that NETs are jointly responsible for cancer-related pathologies like thrombosis, organ failure and metastasis formation
  • NETs might also have a deleterious effect on the host, because the extracellular exposure of histone complexes could play a role during the development of autoimmune diseases like systemic lupus erythematosus
  • NET-associated host damage
  • A small study published in the journal JAMA Cardiology suggested that NETs played a major role in COVID-19 patients who developed ST-elevation myocardial infarctions
  • These observations suggest that NETs might play an important role in the pathogenesis of infectious, inflammatory and thrombotic disorders
  • NETs also have a role in thrombosis and have been associated with stroke
  • NETs possibly contribute to the hypercoagulable state in HIV by trapping platelets, and expressing tissue factor.
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  • Different mechanisms may contribute to direct and indirect platelet activation during sepsis, including platelet activation by the pathogen (86–90), pathogen- and inflammation-driven activation of the endothelium and leukocytes and complement activation-mediated platelet activation (91). The complexity of platelet activation in sepsis suggests the contribution of multiple receptors, making it likely that combined therapy might be required to inhibit platelet activation in sepsis. However, increased bleeding risk in these patients adds another layer of complexity for targeting platelets in septic patients.
  • latelet activation is also associated with increased platelet-neutrophil and platelet-monocyte aggregates in septic patients (84, 85) further potentiating the inflammatory response. Together these results suggest that septic patients' platelets circulate in an activated state, increasing their thrombotic potential
  • In resting platelets, CD62P is stored in the α-granule membrane and becomes exposed on the platelet surface upon activation
  • The ADP receptors, P2Y1 and P2Y12, are differentially involved in pro-thrombotic platelet activation as well as expression of platelet P-selectin in response to various agonists
  • P2Y12 inhibitors are routinely used in the clinics for prevention of thrombotic complications.
  • P2Y Receptors
  • n infectious settings, FcγRIIA mediates immune complex-induced platelet activation or killing of opsonized bacteria
  • Glycoprotein VI (GPVI) GPVI is an immunoreceptor tyrosine-based activation motif (ITAM) receptor that plays a crucial role in the collagen-induced activation and aggregation of platelets. By binding to exposed subendothelial collagen, GPVI mediates the sealing of vascular injuries and ensures integrity of the circulatory system.
  • In platelets NOD2 contributes to platelet activation and is possibly involved in arterial thrombosis during infection
  • NOD1 recognizes d-glutamyl-meso-diaminopimelic acid primarily from gram-negative bacteria, NOD2 detects the muramyl dipeptide (MDP) motif in peptidoglycan from all bacteria. NOD1 is broadly distributed, whereas NOD2 is mainly expressed in innate immune cells (61) and platelets
  • Following activation platelets upregulate CD40L, promoting their interaction with innate immune cells, in particular monocytes. CD40L can be shed by metalloproteinase 9 (MMP9) which is upregulated during sepsis
  • receptors that bind to conserved bacterial structures via carbohydrate-recognition
  • During gram-negative infection but not gram-positive infection, TLR4 activation induces the expression of neuraminidase, promoting alkaline phosphatase clearance and increasing LPS phosphorylation and toxicity
  • TLRs are type I transmembrane proteins
  • Each TLR detects distinct PAMPs and therefore recognizes viruses, bacteria, mycobacteria, fungi, or parasites
  • Toll-Like Receptors (TLRs)
  • Moreover, some receptors are not expressed in mice, while they fulfill important immunomodulatory functions in humans (e.g., FcγRIIA). This makes it often difficult to translate results from animal models to the clinical situation.
  • Women, for example, express more copy numbers of TLRs compared to men (40) and receptor expression correlates with distinct cardiovascular risk and inflammatory biomarkers
  • It is currently unknown if
  • Platelets express various receptors that are involved in the initiation and progression of sepsis. They include receptors for pathogen recognition, immune cell activation and platelet activation.
  • Platelets In Sepsis
  • On the other hand, platelets can inhibit inflammation and promote tissue repair in a receptor- and organ-dependent manner. Therefore, the balance between the pro-inflammatory and anti-inflammatory roles of platelets regulates the outcome.
  • Platelets are also involved in monocyte differentiation into macrophages and modulate their effector functions. Thereby platelets also contribute to excessive inflammatory host response during sepsis and promote the development and progression of sepsis via their involvement in both inflammation and thrombosis.
  • They readily interact with innate immune cells and exert immunomodulatory effects directly via cell-cell contact or indirectly via the release of chemokines and cytokines (37). Platelets promote endothelial adhesion and extravasation of leukocytes at sites of inflammation while securing vascular integrity at the site of transmigratio
  • Platelets are crucial regulators of leukocyte function and thus of inflammatory immune responses
  • A deeper knowledge of the role of platelet receptors in sepsis along with randomized clinical trials will determine the beneficial potential of different anti-platelet therapies in patients.
  • Platelets are not only pro-inflammatory cells but they also contribute to the resolution of inflammation and tissue repair. Most of the studies performed in mice use wild-type mice that lack FcγRIIA on platelets, one of the major receptors on platelets regulating pathogen-mediated activation, raising the question if FcγRIIA transgenic mice are required to investigate infection-mediated sepsis in mice
  • While patients with SIRS would benefit from an anti-inflammatory therapy, immune-suppressed patients might benefit from an immuno-adjuvant therapy
  • Moreover, as platelet receptors regulate inflammatory hemostasis and infection in a stimulus- and organ-dependent manner, a better understanding of the receptors and the mechanisms involved is crucial for successful treatment.
  • Many strategies are currently under investigation to restore platelet count in sepsis patients. However, it is still not known whether thrombocytopenia is a cause or a consequence of sepsis severity and how platelets contribute to sepsis progression.
  • inhibition of cyclooxygenase-1 (COX-1) ameliorates thrombocytopenia and kidney dysfunction in endotoxemia (179), yet transfusion of COX-1-deficient platelets into platelet-depleted mice leads to worse survival than transfusion of wildtype platelets
  • Indeed, the role of platelets may depend on the specific pathologic setting and thus experimental model, as platelet depletion in Streptococcus pyogenes actually ameliorates weight loss, decreases bacterial burden and dampens the inflammatory host response (143). The reason for this discrepancy with other reports is currently unknown.
  • In line with these findings, thrombocytopenia also exacerbates the inflammatory response in sepsis, raising plasma levels of TNF-α, IL-6, IL-10, myeloperoxidase (MPO), monocyte chemotactic protein 1 (MCP-1), and interferon-γ (IFN-γ) (141, 144, 159), potentially as a consequence of more severe infection.
  • low platelet counts are associated with increased secondary hemostasis, liver and kidney damage as well as exacerbated bacteremia and systemic bacterial dissemination in bacteria-induced sepsis
  • Cecal ligation and puncture (CLP) represents one of the most commonly used sepsis models as it most closely resembles sepsis in humans regarding biochemical, hemodynamic and immune responses, including hypotension, leukopenia, thrombocytopenia with a concomitant pro-thrombotic and pro-coagulatory phenotype, raised levels of pro-inflammatory cytokines as well as markers of organ dysfunction
  • Therefore, infection with live bacteria also allows studying of antibacterial host responses including phagocytosis, formation of NETs and release of antimicrobial agents, which play important roles in human sepsis.
  • e.g., pneumosepsis, in which infections originate in the lungs before spreading systemically. Accordingly, intranasal infection with 104-106 colony forming units (CFU) of Klebsiella pneumoniae or Streptococcus pneumoniae, the most common gram-negative and gram-positive causative pathogens of community-acquired pneumonia, respectively (146), induces local pulmonary inflammation with accompanied cytokine response and infiltration of neutrophils and macrophages into the inflamed lungs. As local immunity becomes unable to contain the infection, bacteria disseminate into the bloodstream and can be detected in distant organs such as spleen, kidneys, and liver
  • Additionally, due to the immediate effect on endothelial cells and the vasculature, intravenous administration triggers a potent, rapid, pro-inflammatory immune response that may be stronger than the host response induced by a local infection.
  • Therefore, severity and persistence of thrombocytopenia as well as immature platelet fractions and platelet microvesicle composition are strong predictors of mortality in sepsis.
  • Severe thrombocytopenia is independently associated with disease severity and mortality at the ICU admission and is associated with a dysregulated host response
  • Indeed, the kinetics of platelets in sepsis is often biphasic, characterized by an initial drop within the first few days (day 1–4) followed by an increase in platelets and thrombocytosis (96). Lack of this biphasic response leads to persistent thrombocytopenia and is associated with poor prognosis and increased 28-day mortality
  • However, as platelet receptors play different roles in thrombosis, inflammatory hemostasis and inflammation, precautions have to be taken when targeting platelets in infection
  • Inhibition of platelet function in sepsis represents an attractive target due to their role in thrombosis and inflammation
  • Administration of aspirin for 24 h at the time of SIRS recognition is associated with increased survival in a large cohort of over 5,000 septic patients
  • In human experimental endotoxemia, P2Y12 inhibitors reduce the pro-inflammatory and pro-thrombotic mechanisms
  • Several observational and retrospective clinical studies have shown that anti-platelet agents such as aspirin (COX-1 inhibitor), platelet P2Y12 receptor antagonists like clopidogrel or GPIIb/IIIa antagonists reduce mortality or complications in critically ill patients
  • An increase in the proliferation and activation of monocytes and macrophages in the bone marrow was observed in septic patients with thrombocytopenia. The uncontrolled proliferation is associated with an increase in macrophage-colony stimulating factor (M-CSF) which accelerates the ingestion of hematopoietic cells by macrophages and may contribute to thrombocytopenia
  • Moreover, IgG-opsonization enhances the clearance of LPS-binding platelets in an Fc-dependent manner and further potentiates platelet clearance in gram-negative infection (51). Inflammation fosters immune thrombocytopenia as C-reactive protein, produced during the acute phase of inflammation, enhances antibody-mediated platelet clearance by FcγR-dependent phagocytosis
  • Anti-platelet antibodies (e.g., anti-PF4/heparin) are detected in patients with bacterial septicemia and their level increases in thrombocytopenic patients with no significant difference between gram-positive and gram-negative infection
  • suggesting that platelet activation by pathogens contributes to thrombocytopenia but does not represent a major mechanism
  • Some pathogenic bacteria, particularly blood stream infections, may also trigger apoptosis in platelets resulting in thrombocytopenia (107, 108). Thrombocytopenia occurs in 20–30% of patients infected with Staphylococcus aureus, Escherichia coli, or Streptococcus pneumonia
  • An overview of these processes is given in Figure 2.
  • Causes of Thrombocytopenia in Septic Patients
  • widespread cellular injury, which precedes organ dysfunction. The precise mechanism of cellular injury is highly complex and still incompletely understood. The main mechanisms involved include: cytopathic injury, which is mediated by direct cell injury by pro-inflammatory mediators and/or other products of inflammation, tissue ischemia due to insufficient oxygen supply, and an altered rate of apoptosis
  • Microcirculatory lesions further occur as a result of imbalances in the coagulation and fibrinolytic systems, both of which are activated during sepsis.
  • Tissue ischemia is caused by endothelial and microcirculatory lesions
  • Circulating inflammatory mediators activate the endothelium and induce loosening of tight junctions between endothelial cells, thereby increasing vascular permeability and leakage. As a consequence, systemic endothelial activation leads to hypotension and edema formation and thus to inadequate tissue oxygenation
  • tissue ischemia due to insufficient oxygen supply,
  • While sepsis can be caused by infection with bacteria, virus and fungi, the most frequent pathogens in sepsis are gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae
  • The most common causes of sepsis are infections of the respiratory system, followed by genitourinary and abdominal infections
  • The systemic dissemination of the immune response to uninfected remote tissue and failure to restore homeostasis, leads to a malignant intravascular inflammation called sepsis.
  • A fine-tuned balance of pro-inflammatory and anti-inflammatory mediators regulates and restricts the inflammatory processes, the invading pathogens are cleared, homeostasis is restored and tissue repaired
  • These processes are responsible for the cardinal signs of local inflammation: warmth and erythema due to local vasodilation and hyperemia, protein-rich edema due to increased microvascular permeability and pain due to mediators released by innate immune cells.
  • As first line of defense to infection, the complement cascade, neutrophils and endothelial cells are activated, inducing the expression of adherence molecules on endothelial cells and promoting neutrophil and subsequent monocyte migration and extravasation to the site of inflammation. Monocytes differentiate into macrophages in situ and secrete a mixture of pro-inflammatory [e.g., tumor necrosis factor (TNF)-α and interleukin (IL)-1] and anti-inflammatory (e.g., IL-10) mediators. The ingestion of apoptotic neutrophils by inflammatory macrophages induces their switch to anti-inflammatory macrophages with repair properties
  • Engagement of PRRs elicits various signaling cascades essential for the neutralization of pathogens. A central event in this process is the activation of cytosolic nuclear factor-κB (NF-κB). Activated NF-κB translocates from the cytoplasm to the nucleus, where it binds to transcription sites and induces activation of a plethora of genes involved in inflammatory host response, including pro-inflammatory cytokines, chemokines, adhesion molecules, and nitric oxide (NO) synthase (6).
  • PRRs can be further divided in toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD)-like receptors, and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)
  • released from injured tissues during the inflammatory insult.
  • PRR
  • Microbial components can be recognized by germline-encoded
  • Normal host response to pathogen invasion involves a complex process to localize and confine microbes, while initiating repair processes of injured tissue
  • Sepsis is typically manifested by an early dominant hyper-inflammatory phase, the systemic inflammatory response syndrome (SIRS), characterized by fever and hyper-metabolism, which can eventually lead to septic shock. This pro-inflammatory state is followed by or co-exists with a compensatory anti-inflammatory response (CARS) and immunosuppression, leading to secondary complications (2).
  • organ dysfunction caused by dysregulated inflammatory host response to an overwhelming systemic infection
  • Sepsis is caused by a dysregulated host response to infection, leading to organ dysfunction, permanent disabilities, or death. During sepsis, tissue injury results from the concomitant uncontrolled activation of the complement, coagulation, and inflammatory systems as well as platelet dysfunction. The balance between the systemic inflammatory response syndrome (SIRS) and the compensatory anti-inflammatory response (CARS) regulates sepsis outcome. Persistent thrombocytopenia is considered as an independent risk factor of mortality in sepsis, although it is still unclear whether the drop in platelet count is the cause or the consequence of sepsis severity.
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  • Note lack of obliteration of bronchial and vascular markings and lack of architectural distortion
  • Although it is not pathognomonic, we have observed the crazy paving pattern to be highly characteristic of early active SARS in the appropriate clinical setting. This sign has been observed in 10 (37%) of 27 patients, including four patients showing evidence of clinical relapse.
  • Three patients who had follow-up examinations performed 2 weeks after the first HRCT examination showed complete resolution of the ground-glass opacities.
  • Our observation suggested that among the earliest signs of the disease, ground-glass density and crazy paving pattern are key features. These signs occurred in the first week after onset of symptoms.
  • Each of selected HRCT signs was charted against the mean duration from onset of symptoms
  • Traction bronchiolectasis was defined as bronchiole dilatation, which was commonly irregular, in association with juxtabronchial opacification that was interpreted as representing retractile pulmonary fibrosis
  • Honeycombing was defined as clustered cystic air spaces usually of comparable diameters of 0.3- to 1-cm cysts and as large as 2.5 cm, usually subpleural and characterized by well-defined walls, which were often thick [16]. Subpleural emphysematous blebs were defined as focal thin-walled radiolucency contiguous with the pleura
  • Masslike organizing density was defined as an area of irregular masslike dense shadow obliterating or distorting vessels and bronchi within it and around the lesion.
  • Septal thickening perpendicular to the pleural surface
  • A subpleural line
  • Micronodule
  • Marble shadow was defined as an area of patchwork-like inhomogeneous increase in attenuation without obliteration of underlying vessels.
  • Consolidation with or without air bronchogram was defined as homogeneous density with obliteration of underlying vessels [
  • hick reticular shadows superimposed on background parenchymal opacification
  • Ground-glass opacity was defined as hazy increased attenuation of the lung, with preservation of bronchial and vascular markings
  • The underlying vessels were not obliterated, and there was no architectural distortion.
  • Crazy paving pattern was defined as a thin reticular shadow superimposed on ground-glass opacities that resembled cobblestones
  • Pleural effusion, other complications such as pneumothorax and pneumomediastinum, and other coexisting disease (e.g., tuberculous granuloma, bronchiectasis, emphysema bullae) if any, were also recorded. HRCT findings were correlated with the onset of clinical symptoms. The lobar distribution of the lesions was also recorded.
  • Reticular pattern was defined
  • All HRCT scans were reviewed by two radiologists, with the final impression and diagnosis reached by consensus. We sought HRCT signs with the following working definitions:
  • Our analysis focuses on a pattern-based approach.
  • The timing for initiation of therapy is important because prompt treatment is found to have a better outcome for patients on the basis of early experience in Hong Kong
  • A defined pattern of HRCT findings is observed in different phases of SARS, which is characterized by focal ground-glass and crazy paving patterns in a scattered distribution at presentation, followed by development of interstitial thickening, consolidation, pleural reaction, and scarring. Spontaneous pneumomediastinum is a distinct complication during the course of the illness.
  • Consolidation tends to be associated with eventual formation of masslike organizing density and scars. We have also observed subtle increases in parenchymal background density in affected areas of the lung on follow-up studies in some patients, despite resolution of most other changes (Fig. 12). We are not sure whether this represents diffuse fibrosis at a microscopic level and hence permanent residual lung damage. Volume loss has also been observed.
  • Permanent lung damage is present when fibrous bands, interstitial fibrosis, honeycomb changes and pleural thickening and tethering, or progression of masslike organizing shadows to scars is noted
  • Masslike organizing density, changes similar to massive pulmonary fibrosis with peripheral emphysema in pneumoconiosis, has been observed in patients with SARS.
  • Spontaneous pneumomediastinum is a distinctive complication of SARS in the early reparative phase. Pleural effusion and other forms of pleural reaction have been observed in our patients.
  • Ground-glass opacity and crazy paving pattern in a scattered distribution are typical early changes of the disease. Further progression of disease leads to development of interstitial changes and consolidation, which are followed by a reparative phase with disease resolution or fibrosis and scarring.
  • a fairly defined but broad pattern of change has been observed on HRCT in different phases of SARS.
  • paraseptal emphysema
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  • The authors defined that a child would be anemic if its hemoglobin concentration was below 11 g/dL, regardless of age.
  • Simple upper respiratory infections leading to anemia are quite unusual,
  • But in general this type of infection is more severe.
  • In parvovirosis, on the other hand, anemia is secondary to the inhibition of medullary erythropoiesis caused by this virus.
  • When anemia exists in acute infections, it is due to several factors. Clearly, in a patient with malaria the main reason for anemia is the destruction of red blood cells by the parasite
  • Hepcidin induces the degradation of ferroportin, responsible for the transmembrane transfer of iron to plasma transferrin
  • Although other mechanisms may be operating concurrently, depending on the underlying disease, the main one is impairment in the release of iron by the reticuloendothelial system.
  • his happens, briefly, due to the increased concentration of hepcidin, a regulatory hormone produced by liver cells that rises with inflammation
  • Let us see what is the current understanding for the most frequent cases in which anemia is associated with chronic infections or inflammatory diseases.(2-4) In these cases anemia is linked to reticuloendothelial siderosis, and is usually mild or moderate. It is characterized by decreased serum iron with a decrease in the total iron binding capacity and percentage of transferrin saturation. Serum ferritin may be normal or increased. The differences in the laboratory tests for iron deficiency anemia are that, in the latter, the transferrin binding capacity rises, the ferritin is low and marrow iron is missing.
  • The management of the hematopoietic factor, generally involving intravenous iron, raises the level of hemoglobin, even without changes in other aspects of patient care.
  • In advanced renal failure, although other mechanisms are at work, the main reason for severe anemia is a deficiency in the production of erythropoietin by the damaged kidney tissue
  • Of course there may be other mechanisms that contribute to the onset of anemia, such as blood loss, nutritional deficiencies, side effects of medications and elevations of certain cytokines that are responsible, in most cases of chronic diseases, for the association with anemi
  • leukemia that, by definition, originates in the bone marrow, gradually leading to anemia both because of the progressive occupation of the tissue that produces the red blood cells and due to their normal senescence. The degree of anemia and the speed in the drop of hemoglobin concentrations depend on the different subtypes of leukemia.
  • Supposedly there is a common pathophysiological basis for the occurrence of anemia and several chronic diseases, whether infectious, inflammatory or neoplastic.
  • When it comes to chronic infection
  • Many other acute infections, either viral or bacterial, can cause anemia through other mechanisms, such as mild idiopathic hemolysis and marrow inhibition.
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  • Mechanisms of pathogen clearance by platelets may be direct, through the release of various antimicrobial peptides and indirect via the release of platelet-derived mediators that coordinate chemotaxis and activation of immune cells [83, 116,117,118, 120, 121]. Infection is commonly associated with tissue injury. Injured and dying cells generate mediators such as alarmins that fuel inflammation [122]. Mediators generated by cell damage such as complement activation products and histones can activate platelets [123, 124]. Notwithstanding, platelets also contribute to the adaptive immune response to infection [17, 18, 22, 125].
  • Platelet interaction with bacteria depends on the nature and concentration of bacteria, interaction time, and involves multiple mechanisms.
  • PMPs are also thought to contribute to vascular repair [115]. Hence, platelet activation is both necessary to tissue integrity and undesirable as it generates tissue-damaging signals
  • Platelets have however been found to both promote and prevent vascular permeability in inflammation. The differential regulation of vascular permeability by platelets has been studied for a large part in acute lung injury (ALI) models and will be presented in the corresponding section.
  • Platelets provide material for endothelium repair, including EC growth-promoting, antiapoptotic mediators, and attractants for progenitor cells endowed with vascular healing properties [104]. They help restoring the disrupted vascular network, providing positive and negative regulators of angiogenesis and stimulating angiogenic mediator production by target cells.
  • The best studied role of platelets in tissue homoeostasis is the preservation of resting and injured endothelium integrity
  • First, platelets support the generation of thrombin. Second, platelet links inflammation and coagulation. Third, platelets are major inducers of the release of pro-thrombotic scaffolds neutrophil extracellular trap
  • Platelet inflammatory mediators may thus contribute to sepsis coagulopathy [88,89,90,91]. DIC is a frequent and major complication of sepsis [41], and various mechanisms concur to involve platelets in DIC
  • The activation of coagulation and inflammation cascades are consequences of platelet activation, and inflammation and coagulation pathways crosstalk [84]. For example, some platelet mediators have both inflammatory and pro-coagulant properties, such as polyphosphates
  • Importantly, they also contribute to the control and resolution of inflammation via several mechanisms, including the release of anti-inflammatory cytokines and inflammation pro-resolving mediators
  • platelets activate neutrophils and monocytes upon interaction, via several mechanisms, including the triggering of TREM-1 on neutrophils, leading to various pro-inflammatory responses
  • Platelet/leucocyte interactions are a critical step in leucocyte recruitment, activation and migration in inflammation
  • these effects have to be paralleled with the opposed protective role of platelets (below) [66,67,68,69]. Leucocytes are a second critical target for platelets, the platelet/leucocyte dialogue being essential in inflammation; here, we focus on neutrophils and monocytes.
  • Platelet activation in inflammation can alter the vascular tone and lead to deleterious effects on vasculature integrity, by increasing vascular barrier permeability and contributing to the generation of cytopathic signals, for example by mediating reactive oxygen species generation by neutrophils
  • Platelets adhere to activated ECs, following a multi-step process in which glycans play a critical role
  • ECs and leucocytes are prime targets for platelets
  • Activated platelets secrete a profusion of pro-inflammatory material, cytokines/chemokines, vasoactive amines, eicosanoids, and components of proteolytic cascades that directly or indirectly, through the activation of bystander target cells, fuel inflammation
  • Platelet-derived microparticles (PMPs) recapitulate several of activated platelet functions
  • Platelets are activated in conditions that disrupt tissue homoeostasis and exert, directly and indirectly, a complex control over the different stages of inflammation, contributing to pathogen clearance, wound repair and tissue regeneration
  • The archetypal function of platelets is haemostasis. Platelets encounter inhibitory signals that prevent their activation in the healthy vasculature, such as nitric oxide and prostacyclin, which are released by endothelial cells (ECs). Platelets circulate in close proximity to the vessel wall, and the disruption of EC lining overcomes inhibitory signals and drives platelet adherence, activation and aggregation, which temporarily plug the damaged vessel. In this process, platelets also activate and confine coagulation at site of damage
  • In pathological conditions associated with platelet activation, multiple agonists are generated. In fact, apart from classical strong agonists such as thrombin or collagen, there is an expanding list of agonists that can contribute to platelet activation. These additional platelet agonists have allowed a re-appreciation of mechanisms and role of platelet activation in vascular inflammation and thrombotic events associated with a range of infectious and inflammatory conditions
  • The secretion of granule content following platelet activation by agonists is central to platelet functions
  • maintains numbers of 150,000–400,000 platelets per microlitre of blood
  • Platelets are small (2–4 μm), anucleate, discoid-shaped cytoplasmic fragments
  • Platelets have a short lifespan, of up to 10 days. They are cleared out from the circulation by mechanisms involving lectin–carbohydrate recognition by splenic and liver macrophages and hepatocytes
  • Interestingly, mechanisms of non-septic systemic-associated inflammatory response syndrome (SIRS) as met in major surgery, severe trauma, extensive burns or pancreatitis may share common features with sepsis-associated SIRS, taking the form of a comparable early inflammatory storm that is triggered by alarmins released by damaged tissues
  • The friend and foe dialogue between platelets and endothelium has been extensively studied and is thought to be relevant to sepsis complications.
  • The matter is a complex one as platelets are not only vectors of inflammation contributing to vascular and tissue injury in acute or chronic inflammation [18, 22, 23], but also play an important role in the resolution of inflammation, vascular protection and the repair of damaged tissues.
  • latelets are now acknowledged as essential actors of the immune response, reacting to infection and disturbed tissue integrity and contributing to inflammation, pathogen killing and tissue repair
  • Third, beyond the confines of haemostasis and thrombosis,
  • Sepsis is a syndrome based on a dysregulated immune response to infection also involving non-immunologic mechanisms, including neuroendocrine, cardiovascular and metabolic pathways
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  • Growth Factors
  • Granulocyte colony-stimulating factors (G-CSFs) and granulocyte-macrophage colony-stimulating factors that are available include:
  • Antibiotics
  • the risk of developing a bacterial infection increases significantly.
  • below 1,000 cells/uL
  • especially with regard to neutrophils. The range for absolute neutrophil count is between 2,500 cells/uL and 6,000 cells/uL.
  • Absolute Neutrophil Count
  • Total White Blood Cell Count
  • Red blood cell indices (such as MCV) can sometimes give important clues as to causes such as vitamin B12 deficiency
  • From a prognostic standpoint, recent research suggests that Lymphopenia predicts the severity of disease, and likelihood that it will progress to the need for intensive care or death with COVID-19.7
  • Some viral infections
  • Corticosteroids
  • Lymphopenia: Lymphopenia without a correspondingly low level of other white blood cells is not very common but can be very important
  • Allergic conditions, such as hives (urticaria), severe allergies, angioedema, and anaphylaxis
  • Basopenia:
  • During the acute phase of infections or inflammation
  • Eosinopenia also appears to be an important marker for sepsis
  • whereas conditions involving the bone marrow usually affect all types of white blood cells.
  • due to the risk of infection. Neutropenia without general leukopenia (isolated neutropenia) suggests causes such as autoimmune diseases or vitamin deficiencies
  • Neutropenia:
  • Causes of Low Levels Specific Types of White Blood Cells
  • Vitamin B12 and folate deficiencies are a relatively common cause, as well as iron deficiency anemia.
  • Several of these infections may also cause anemia (a low red blood cell count) and thrombocytopenia (a low platelet count)
  • Bacterial infections
  • There are some infections in which leukopenia is quite common, including: Viral infections
  • With sepsis, an overwhelming body-wide bacterial infection, leukopenia may occur as available white blood cells are "used up" fighting the infection.
  • Leukopenia may occur during the acute infection with some infections or primarily in the postinfectious stage with others.
  • Infections are, counterintuitively, a relatively common cause of leukopenia.
  • Heart medications, such as thiazide diuretics, beta-blockers, and spironolactone
  • Psychiatric medications such as clozapine,
  • Antivirals such as acyclovir
  • non-steroidal anti-inflammatory drugs such as ibuprofen
  • penicillin derivatives (such as Amoxicillin), cephalosporins,
  • Antibiotics,
  • Other Medications:
  • the point at which the white blood cell count reaches its lowest point (the nadir) is roughly 7 to 14 days after an infusion.
  • A low white blood cell count due to chemotherapy
  • If thrombocytopenia s also present, signs may include: Bruising Small red spots on the skin that don't blanch with pressure (petechiae) Nosebleeds Blood in the urine or stool Heavy menstrual periods
  • If anemia (a low red blood cell count) also occurs, symptoms may include: Lightheadedness or fainting A rapid heart rate Pale skin
  • It's important to note that, even when a serious infection is present, signs and symptoms may not be as apparent due to the lack of white blood cells. (White blood cells are responsible for creating the signs of inflammation, pus, etc.)
  • Symptoms of infection may include: Fever, chills, and/or night sweats Headache or stiff neck Sore throat Mouth sores or white patches in the mouth Cough or shortness of breath Pain or burning with urination Drainage, redness, or swelling around a skin wound Abdominal pain and/or diarrhea
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  • The next step is to determine what type of infection you have: bacterial or viral (no guessing allowed)
  • An increase in one type of white blood cell can cause a decrease in other types of white blood cells. For example, if you have a bacterial infection, you will have an increase of neutrophils and a decrease in lymphocytes. Conversely, if you have a viral infection, you will have a decrease in neutrophils and an increase in lymphocytes.
  • Neutrophils are by far the most common form of white blood cells that you have in your body (pus is simply dead neutrophils). Neutrophils are infection fighters that increase during bacterial infections (neutrophils are also known as granulocytes (grans), polys, PMNs, or segs). Lymphocytes, on the other hand, can increase in cases of viral infections.
  • The five types of white blood cells, also called leukocytes, appear in the blood: Neutrophils, Lymphocytes (B cells and T cells), Monocytes, Eosinophils, and Basophils.
  • A normal white blood cell count is in the range of 4,000 to 11,000 cells per liter of blood. If you have an elevated white blood cell count (anything above 11,000), it’s a good bet you have an infection
  • How can you tell whether you have a bacterial or viral infection?
  • If you have a viral infection, you need to drink a lot of fluids and rest until the virus runs its course over 7-10 days.
  • Bacteria are actually useful to the body as well; only about 10% of the bacteria cause harm to the human body. You have millions of bacteria in our intestines and they help digest foods, but they are harmful once they get into the bloodstream. The job of your immune system is to protect your body from these infections.
  • Viral illnesses may cause body aches and fever but usually run their course in 7-10 days
  • more serious concerns are bacterial illnesses like sepsis (bacteria in the blood), bacterial meningitis (bacterial infection in the lining of the brain and spinal cord), bacterial endocarditis (bacteria in the lining of the heart), brain abscess (a pus collection within the brain)
  • Some ailments, such as pneumonia, meningitis, and diarrhea, can be caused by either type of pathogen (dangerous microscopic organisms).
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  • After incubation with Spike or S1 protein, platelets also displayed markedly accelerated spreading (Fig. ​(Fig.4c)4c) and clot retraction (Fig. ​
  • These data indicate that S1, but not S2, binds ACE2 to regulate platelet function, which corroborates the finding that the receptor-binding domain (RBD) of the Spike protein is found in the S1 subunit
  • SARS-CoV-2 virus directly induces decrease in platelet ACE2
  • fluorescent confocal microscopy and TEM revealed that SARS-CoV-2 particles were present inside the platelets (Fig. ​(Fig.3e,3e, g), suggesting that SARS-CoV-2 can infect these cells.
  • SARS-CoV-2 virus directly potentiates platelet activation
  • We found that human platelets exhibit robust expression of ACE2 at both the RNA and protein levels as detected by RT-PCR (Fig. ​(Fig.2(A1))2(A1)) and Western blot (Fig. ​(
  • We found that human platelets exhibit robust expression of ACE2 at both the RNA and protein levels as detected by RT-PCR (Fig. ​
  • platelet-poor plasma (PPP)
  • SARS-COV-2 RNA-positive PPP enhanced platelet aggregation, compared with SARS-COV-2 RNA-negative PPP and healthy PPP
  • notably, PAC-1 binding and CD62P expression were both moderately correlated with decreases in platelet count (Pearson │r│ for PAC-1 = 0.50, P < 0.01; Pearson │r│ for CD62 = 0.64, P < 0.01; Fig. ​Fig.1i)1i) in COVID-19 patients.
  • Platelet activation leads to platelet consumption which causes thrombocytopenia
  • Consistent with the increased MPV, we found that integrin αIIbβ3 activation (PAC-1 binding) and P-selectin (CD62P) expression were increased in platelets of COVID-19 patients
  • The relationship between the platelet count and coagulation parameters in COVID-19 patients is described in Fig. ​Fig.1a–f.1a–f. For those with a normal platelet count (> 125 × 109/L), the platelet count did not significantly impact the outcomes of the PT, PTA, INR, APTT, d-dimer, and FDPs tests. However, in patients with thrombocytopenia (< 125 × 109/L), when the platelet count decreases, PT, INR, APTT, d-dimer, and FDPs increase exponentially, while PTA decreases exponentially. We further examined platelet count over time in severe and critically severe COVID-19 patients (n = 22). Our results suggest that platelet count decreases gradually after hospital admission (Fig. ​(Fig.11g).
  • MPV was shown to correlate with platelet activity and is considered a marker of platelet activity [49, 50]; therefore, increased MPV in COVID-19 suggests that these platelets may present as hyperactive.
  • Severe and critically severe COVID-19 patients presented with abnormal platelet parameters, including decreased platelet counts and plateletcrit (PCT), increased mean platelet volume (MPV), and platelet distribution width (PDW) as well as abnormal coagulation parameters including increased prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), d-dimer and fibrinogen degradation products (FDPs), and decreased prothrombin time activity (PTA) when compared with healthy donors, non-COVID-19 patients, and mild and moderate COVID-19 patients (Additional file 1: Online Table 3
  • Moreover, we were able to demonstrate that SARS-CoV-2 and its Spike protein directly stimulate platelets resulting in coagulation factor release, inflammatory cytokine secretion, and leukocyte–platelet aggregates (LPAs) formation. Finally, we provide evidence that treatment with recombinant human ACE2 protein and an anti-Spike monoclonal antibody can reverse SARS-CoV-2 Spike protein-induced platelet activation.
  • Here, we report that platelets from COVID-19 patients are hyperactive, and demonstrate, for the first time, that platelets express ACE2 and TMPRSS2. SARS-CoV-2 and its Spike protein directly bind platelet ACE2 and enhance platelet activation in vitro. The Spike protein also potentiates thrombus formation in vivo.
  • SARS-CoV-2 uses its Spike protein to enter host cells by binding to angiotensin-converting enzyme 2 (ACE2) on the host cell membrane [23–26]. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a serine protease, proteolytically cleaves and activates the Spike protein to facilitate SARS-CoV-2 virus-cell membrane fusions.
  • During infection, activated platelets adhere to the sub-endothelium, and their hyperactivity results in thrombus formation, leading to arterial ischemia and even pulmonary embolisms. Many viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, Ebola, and Dengue virus (DV), can directly lead to platelet hyperactivity [13–16]. Influenza virus directly activates platelets and triggers uncontrolled coagulation cascades and consequent lung injur
  • We investigated ACE2 expression and direct effect of SARS-CoV-2 virus on platelets by RT-PCR, flow cytometry, Western blot, immunofluorescence, and platelet functional studies in vitro, FeCl3-induced thrombus formation in vivo, and thrombus formation under flow conditions ex vivo.
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