Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis.

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Relative Hypercoagulopathy of the SARS-CoV-2 Beta and Delta Variants when Compared to the Less Severe Omicron Variants Is Related to TEG Parameters, the Extent of Fibrin Amyloid Microclots, and the Severity of Clinical Illness.

Earlier variants of SARS-CoV-2 have been associated with hypercoagulability and an extensive formation of fibrin amyloid microclots, which are considered to contribute to the pathology of the coronavirus 2019 disease (COVID-19). The newer omicron variants appear to be far more transmissible, but less virulent, even when taking immunity acquired from previous infections or vaccination into account. We here show that while the clotting parameters associated with omicron variants are significantly raised over those of healthy, matched controls, they are raised to levels significantly lower than those seen with more severe variants such as beta and delta. We also observed that individuals infected with omicron variants manifested less extensive microclot formation in platelet-poor plasma compared with those harboring the more virulent variants. The measurement of clotting effects between the different variants acts as a kind of "internal control" that demonstrates the relationship between the extent of coagulopathies and the virulence of the variant of interest. This adds to the evidence that microclots may play an important role in reflecting the severity of symptoms observed in COVID-19.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-Acute Sequelae of COVID-19 (PASC).

BACKGROUND: Fibrin(ogen) amyloid microclots and platelet hyperactivation previously reported as a novel finding in South African patients with the coronavirus 2019 disease (COVID-19) and Long COVID/Post-Acute Sequelae of COVID-19 (PASC), might form a suitable set of foci for the clinical treatment of the symptoms of Long COVID/PASC. A Long COVID/PASC Registry was subsequently established as an online platform where patients can report Long COVID/PASC symptoms and previous comorbidities. METHODS: In this study, we report on the comorbidities and persistent symptoms, using data obtained from 845 South African Long COVID/PASC patients. By using a previously published scoring system for fibrin amyloid microclots and platelet pathology, we also analysed blood samples from 80 patients, and report the presence of significant fibrin amyloid microclots and platelet pathology in all cases. RESULTS: Hypertension, high cholesterol levels (dyslipidaemia), cardiovascular disease and type 2 diabetes mellitus (T2DM) were found to be the most important comorbidities. The gender balance (70% female) and the most commonly reported Long COVID/PASC symptoms (fatigue, brain fog, loss of concentration and forgetfulness, shortness of breath, as well as joint and muscle pains) were comparable to those reported elsewhere. These findings confirmed that our sample was not atypical. Microclot and platelet pathologies were associated with Long COVID/PASC symptoms that persisted after the recovery from acute COVID-19. CONCLUSIONS: Fibrin amyloid microclots that block capillaries and inhibit the transport of O2 to tissues, accompanied by platelet hyperactivation, provide a ready explanation for the symptoms of Long COVID/PASC. Removal and reversal of these underlying endotheliopathies provide an important treatment option that urgently warrants controlled clinical studies to determine efficacy in patients with a diversity of comorbidities impacting on SARS-CoV-2 infection and COVID-19 severity. We suggest that our platelet and clotting grading system provides a simple and cost-effective diagnostic method for early detection of Long COVID/PASC as a major determinant of effective treatment, including those focusing on reducing clot burden and platelet hyperactivation.

TEG®, Microclot and Platelet Mapping for Guiding Early Management of Severe COVID-19 Coagulopathy.

An important component of severe COVID-19 disease is virus-induced endothelilitis. This leads to disruption of normal endothelial function, initiating a state of failing normal clotting physiology. Massively increased levels of von Willebrand Factor (VWF) lead to overwhelming platelet activation, as well as activation of the enzymatic (intrinsic) clotting pathway. In addition, there is an impaired fibrinolysis, caused by, amongst others, increased levels of alpha-(2) antiplasmin. The end result is hypercoagulation (proven by thromboelastography® (TEG®)) and reduced fibrinolysis, inevitably leading to a difficult-to-overcome hypercoagulated physiological state. Platelets in circulation also plays a significant role in clot formation, but they themselves may also drive hypercoagulation when they are overactivated due to the interactions of their receptors with the endothelium, immune cells or circulating inflammatory molecules. From the literature it is clear that the role of platelets in severely ill COVID-19 patients has been markedly underestimated or even ignored. We here highlight the value of early management of severe COVID-19 coagulopathy as guided by TEG®, microclot and platelet mapping. We also argue that the failure of clinical trials, where the efficacy of prophylactic versus therapeutic clexane (low molecular weight heparin (LMWH)) were not always successful, which may be because the significant role of platelet activation was not taken into account during the planning of the trial. We conclude that, because of the overwhelming alteration of clotting, the outcome of any trial evaluating an any single anticoagulant, including thrombolytic, would be negative. Here we suggest the use of the degree of platelet dysfunction and presence of microclots in circulation, together with TEG®, might be used as a guideline for disease severity. A multi-pronged approach, guided by TEG® and platelet mapping, would be required to maintain normal clotting physiology in severe COVID-19 disease.

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. METHODS: We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. RESULTS: We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. CONCLUSIONS: Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by unprecedented clinical pathologies. One of the most important pathologies, is hypercoagulation and microclots in the lungs of patients. Here we study the effect of isolated SARS-CoV-2 spike protein S1 subunit as potential inflammagen sui generis. Using scanning electron and fluorescence microscopy as well as mass spectrometry, we investigate the potential of this inflammagen to interact with platelets and fibrin(ogen) directly to cause blood hypercoagulation. Using platelet-poor plasma (PPP), we show that spike protein may interfere with blood flow. Mass spectrometry also showed that when spike protein S1 is added to healthy PPP, it results in structural changes to ß and γ fibrin(ogen), complement 3, and prothrombin. These proteins were substantially resistant to trypsinization, in the presence of spike protein S1. Here we suggest that, in part, the presence of spike protein in circulation may contribute to the hypercoagulation in COVID-19 positive patients and may cause substantial impairment of fibrinolysis. Such lytic impairment may result in the persistent large microclots we have noted here and previously in plasma samples of COVID-19 patients. This observation may have important clinical relevance in the treatment of hypercoagulability in COVID-19 patients.

Psoriatic disease is associated with systemic inflammation, endothelial activation, and altered haemostatic function.

Psoriasis is a chronic, immune-mediated inflammatory skin disease, affecting approximately 2% of the general population, which can be accompanied by psoriatic arthritis (PsA). The condition has been associated with an increased cardiovascular burden. Hypercoagulability is a potential underlying mechanism that may contribute to the increased risk of major cardiovascular events in psoriatic individuals. Whole blood samples were collected from 20 PsA patients and 20 healthy individuals. The concentrations of inflammatory molecules (C-reactive protein, serum amyloid A, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and soluble P-selectin) were determined by enzyme-linked immunosorbent assays. In addition, clotting efficiency was evaluated by thromboelastography. The fibrin network architecture was also assessed by scanning electron microscopy. Elevated levels of circulating inflammatory molecules were significantly associated with the presence of psoriatic disease. Furthermore, an increased tendency towards thrombus formation was significantly predictive of disease presence. Scanning electron microscopy revealed that fibrin clots were denser in psoriatic individuals, compared to healthy controls, with an increased fibrin fibre diameter associated with psoriatic disease. Our results add to the accumulating evidence of the systemic nature of psoriasis and the subsequent risk of cardiovascular comorbidities, potentially due to an acquired hypercoagulability. We suggest that haemostatic function should be monitored carefully in psoriatic patients that present with severe disease, due to the pre-eminent risk of developing thrombotic complications.

The Value of Detecting Pathological Changes During Clot Formation in Early Disease Treatment-Naïve Breast Cancer Patients.

Breast cancer (BC) is one of the most prevalent forms of cancer in women worldwide. Clinical research indicates that BC patients are at an increased risk for thrombotic events, drastically decreasing their quality-of-life and treatment outcomes. There is ample evidence of this in the literature, but it is mainly focused on metastatic BC. Therefore, coagulopathies of nonmetastatic BC are understudied and require in-depth investigation. In this study, clot kinetics and ultrastructure were used to investigate treatment-naïve, nonmetastatic BC patients using scanning electron microscopy, Thromboelastography®, and confocal laser scanning microscopy. It was demonstrated that nonmetastatic BC patients exhibit minimal ultrastructural alterations of the clot components and no changes in the clot kinetics. However, BC patients presented changes to fibrinogen protein structure, compared to matched controls, using an amyloid-selective stain. Together, these findings suggest that coagulation dysfunction(s) in BC patients with early disease manifest at the microlevel, rather than the macrolevel. This study presents novel insights to a method that are more sensitive to coagulation changes in this specific patient group, emphasizing that the coagulation system may react in different forms to the disease, depending on the progression of the disease itself.

Novel arsenic hyper-resistant bacteria from an extreme environment, Crven Dol mine, Allchar, North Macedonia.

Novel hyper-resistant bacteria were isolated from the Crven Dol mine (Allchar, North Macedonia), arsenic-rich extreme environment. Bacteria were recovered from a secondary mineral mixture, an alteration of hydrothermal realgar rich in arsenates (pharmacolite, hornesite, and talmessite). The sample was recovered from the dark part of the mine at 28 m depth. Three bacterial strains and a bacterial consortium were isolated for their capacity to survive exposure to 32 g/L (209 mM) of arsenite, and 176 g/L (564 mM) of arsenate. The 16S rRNA gene analysis identified bacterial isolates as Stenotrophomonas sp. and two Microbacterium spp. This analysis also revealed that bacterial consortium comprise two Bacteriodetes exhibiting similarity to Olivibacter ginsengisoli and to uncultured bacterium, and one γ-proteobacteria with similarity to Luteimonas sp. Among all isolates Stenotrophomonas sp. exhibited the highest tolerance to As compound as well as the capacity to accumulate As inside the cells. Analysis of genes involved in As-resistance showed that recovered isolates possess the genes encoding the ArsB, Acr3(1) and Acr3(2) proteins, indicating that at least a part of their resistance could be ascribed to As-efflux systems described in isolates obtained from human-polluted environments.

Prevalence of readily detected amyloid blood clots in 'unclotted' Type 2 Diabetes Mellitus and COVID-19 plasma: a preliminary report.

BACKGROUND: Type 2 Diabetes Mellitus (T2DM) is a well-known comorbidity to COVID-19 and coagulopathies are a common accompaniment to both T2DM and COVID-19. In addition, patients with COVID-19 are known to develop micro-clots within the lungs. The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated clotting severity and the extent of clotting pathologies in the individual who was infected or not would be highly desirable. METHODS: Platelet poor plasma (PPP) was collected and frozen. On the day of analysis, PPP samples were thawed and analysed. We show here that microclots can be detected in the native plasma of twenty COVID-19, as well as ten T2DM patients, without the addition of any clotting agent, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. Results were compared to ten healthy age-matched individuals. RESULTS: In COVID-19 plasma these microclots are significantly increased when compared to the levels in T2DM. CONCLUSIONS: This fluorogenic test may provide a rapid and convenient test with 100% sensitivity (P < 0.0001) and is consistent with the recognition that the early detection and prevention of such clotting can have an important role in therapy.

Erythrocyte, Platelet, Serum Ferritin, and P-Selectin Pathophysiology Implicated in Severe Hypercoagulation and Vascular Complications in COVID-19.

Progressive respiratory failure is seen as a major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection. Relatively little is known about the associated morphologic and molecular changes in the circulation of these patients. In particular, platelet and erythrocyte pathology might result in severe vascular issues, and the manifestations may include thrombotic complications. These thrombotic pathologies may be both extrapulmonary and intrapulmonary and may be central to respiratory failure. Previously, we reported the presence of amyloid microclots in the circulation of patients with coronavirus disease 2019 (COVID-19). Here, we investigate the presence of related circulating biomarkers, including C-reactive protein (CRP), serum ferritin, and P-selectin. These biomarkers are well-known to interact with, and cause pathology to, platelets and erythrocytes. We also study the structure of platelets and erythrocytes using fluorescence microscopy (using the markers PAC-1 and CD62PE) and scanning electron microscopy. Thromboelastography and viscometry were also used to study coagulation parameters and plasma viscosity. We conclude that structural pathologies found in platelets and erythrocytes, together with spontaneously formed amyloid microclots, may be central to vascular changes observed during COVID-19 progression, including thrombotic microangiopathy, diffuse intravascular coagulation, and large-vessel thrombosis, as well as ground-glass opacities in the lungs. Consequently, this clinical snapshot of COVID-19 strongly suggests that it is also a true vascular disease and considering it as such should form an essential part of a clinical treatment regime.

Effects of Cigarette Smoke Condensate on Growth and Biofilm Formation by Mycobacterium tuberculosis.

MATERIALS AND METHODS: The planktonic and biofilm-forming cultures were prepared in Middlebrook 7H9 and Sauton broth media, respectively, using Mtb strain, H37Rv. The effects of CSC at concentrations of 0.05-3.12 mg/L on growth, biofilm formation and structure were evaluated using microplate Alamar Blue assay, spectrophotometric procedure and scanning electron microscopy (SEM), respectively. Involvement of reactive oxygen species in CSC-mediated biofilm formation was investigated by including catalase in biofilm-forming cultures. RESULTS: CSC did not affect the growth of planktonic bacteria, but rather led to a statistically significant increase in biofilm formation at concentrations of 0.4-3.12 mg/L, as well as in the viability of biofilm-forming bacteria at CSC concentrations of 0.2-1.56 mg/L. SEM confirmed an agglomerated biofilm matrix and irregular bacterial morphology in CSC-treated biofilms. Inclusion of catalase caused significant attenuation of CSC-mediated augmentation of biofilm formation by Mtb, implying involvement of oxidative stress. These findings demonstrate that exposure of Mtb to CSC resulted in increased biofilm formation that appeared to be mediated, at least in part, by oxidative stress, while no effect on planktonic cultures was observed. CONCLUSION: Smoking-related augmentation of biofilm formation by Mtb may contribute to persistence of the pathogen, predisposing to disease reactivation and counteracting the efficacy of antimicrobial chemotherapy.

Gingipain R1 and Lipopolysaccharide From Porphyromonas gingivalis Have Major Effects on Blood Clot Morphology and Mechanics.

Background:Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation. Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots. Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting. Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.

Histological analysis of the effects of cadmium, chromium and mercury alone and in combination on the spleen of male Sprague-Dawley rats.

The mining sector in South Africa is expected to be the fifth largest in the world. Both mining and transport are the most common reasons for an increased risk of human exposure to heavy metal contamination in South Africa. Due to increasing amounts of metals in the environment, this study identified three metals cadmium, chromium and mercury based on the risk of exposure in South Africa. The aim of this study was to investigate the changes in the morphology of the spleen tissue of male Sprague-Dawley rats exposed to these metals alone and in combination by using light microscopy. Forty eight animals in eight experimental groups were exposed, via oral gavage, to these metals at 1000× the World Health Organization's acceptable water limits of each respective metal, alone and in combination, for 28 days. Changes in the histological structure of the spleen were observed using haematoxylin and eosin and picrosirius red staining. Necrosis was observed in all the groups, with the severity varying between the different exposure groups, alone or in combination. Fibrosis in the spleen tissue was only seen in the experimental groups exposed to cadmium and mercury respectively, as well as in the combination of cadmium and mercury.

Induction of hepatic portal fibrosis, mitochondria damage, and extracellular vesicle formation in Sprague-Dawley rats exposed to copper, manganese, and mercury, alone and in combination.

Increased anthropogenic activity and subsequent environmental exposure to heavy metals induce the production of reactive oxygen species (ROS), which increases oxidative stress and the risk of associated diseases. The aim of this study, in a subacute model of toxicity, was to investigate the effects of copper (Cu), manganese (Mn), and mercury (Hg) alone and in combination on the liver tissue of male Sprague-Dawley rats, exposed orally to 100 times the World Health Organization's acceptable water limits of each metal. General histological alterations as well as ultrastructural changes were investigated using light microscopy and transmission electron microscopy (TEM) respectively. Exposure to Cu, Mn, and Hg, alone and in combinations, caused hydropic swelling of the hepatocytes, dilation of the sinusoids, formation of binucleated hepatocytes with an increased inflammatory cell accumulation at the portal triad. Increased collagen deposition with associated fibrosis was also observed. Evaluation of hepatocyte ultrastructure revealed mitochondrial membrane damage and inner membrane swelling especially for hepatocytes exposed to Mn. Extracellular vesicle (EV) formation was observed in the liver tissue of all exposed rats. Furthermore, increased damage observed for metal combinations was possibly due to synergism. In conclusion, Cu, Mn, and Hg alone and as part of a mixture cause cellular damage, inflammation, and fibrosis increasing the risk of associated diseases.

Detection of Citrullinated Fibrin in Plasma Clots of Rheumatoid Arthritis Patients and Its Relation to Altered Structural Clot Properties, Disease-Related Inflammation and Prothrombotic Tendency.

Aims: The risk of cardiovascular events in patients with Rheumatoid Arthritis (RA) is disproportionately heightened as a result of systemic inflammation. The relative effect of autoimmune-associated citrullination on the structure and thrombotic potential of fibrin(ogen) remains unknown. We therefore compared indices of vascular function, inflammation, coagulation and fibrin clot composition in RA patients with healthy controls and evaluated parameter association with disease presence. Methods: Blood samples were collected from 30 RA patients and 30 age- and gender-matched healthy volunteers. Levels of serum amyloid A (SAA), c-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) was measured using a sandwich immunoassay. Whole blood coagulation was assessed using Thromboelastography (TEG®). Fibrin clot networks and fiber structure was investigated using Scanning Electron Microscopy. The detection and quantification of citrullination in formed fibrin clots was performed using a fluorescently labeled Citrulline monoclonal antibody with Fluorescence Wide Field Microscopy. Results: Concentrations of SAA, CRP and ICAM-1 were significantly elevated in RA patients compared to controls. TEG parameters relating to coagulation initiation, rate of fibrin cross-linking, and time to reach maximum thrombus generation were attenuated in RA patients. Microscopic analysis revealed denser networks of thicker fibrin fibers in RA patients compared to controls and multiple citrullinated regions within fibrin clot structures in RA patients were present. Conclusion: Our findings provide novel evidence for the citrullination of fibrin within vasculature is more prominent in RA plasma compared to control plasma and plasma is more accessible than synovial fluid. Citrullinated fibrinogen could play a role as a determinant of thrombotic risk in RA patients.

Oral exposure to cadmium and mercury alone and in combination causes damage to the lung tissue of Sprague-Dawley rats.

Environmental presence and human exposure to heavy metals in air and cigarette smoke has led to a worldwide increase in respiratory disease. The effects of oral exposure to heavy metals in liver and kidney structure and function have been widely investigated and the respiratory system as a target is often overlooked. The aim of the study was to investigate the possible structural changes in the lung tissue of Sprague-Dawley rats after oral exposure for 28 days to cadmium (Cd) and mercury (Hg), alone and in combination at 1000 times the World Health Organization's limit for each metal in drinking water. Following exposure, the general morphology of the bronchiole and lungs as well as collagen and elastin distribution was evaluated using histological techniques and transmission electron microscopy. In the lungs, structural changes to the alveoli included collapsed alveolar spaces, presence of inflammatory cells and thickening of the alveolar walls. In addition, exposure to Cd and Hg caused degeneration of the alveolar structures resulting in confluent alveoli. Changes in bronchiole morphology included an increase in smooth muscle mass with luminal epithelium degeneration, detachment and aggregation. Prominent bronchiole-associated lymphoid tissue was present in the group exposed to Cd and Hg. Ultrastructural examination confirmed the presence of fibrosis where in the Cd exposed group, collagen fibrils arrangement was dense, while in the Hg exposed group, additional prominent elastin was present. This study identified the lungs as target of heavy metals toxicity following oral exposure resulting in cellular damage, inflammation and fibrosis and increased risk of respiratory disease where Hg showed the greatest fibrotic effect, which was further, aggravated in combination with Cd.

Survival of emerging pathogen Acinetobacter baumannii in water environment exposed to different oxygen conditions.

Bacterium Acinetobacter baumannii is a leading cause of hospital infections. Over the last decade, its occurrence in natural environments outside hospital settings has been reported. The aim was to examine the survival of A. baumannii in water media exposed to different ranges of oxygen supply in order to predict its behaviour in the environment. The abundance of five A. baumannii isolates was monitored in nutrient-depleted and nutrient-rich water media in aerated, intermediate and anaerobic conditions (oxygen saturation 96, 56 and 0%, respectively). A. baumannii survived in both media in all tested oxygen concentrations for 50 days. In nutrient-rich water survival of A. baumannii was lowest in anaerobic conditions, while in nutrient-depleted water there was no difference in survival regardless of oxygen availability. A. baumannii formed translucent small colony variants as the fast response (after 1 day) and dormant cells as the prolonged response (after 14 days) to anaerobic conditions. Transmission electron microscopy (TEM) images showed the outer membrane of coccobacillus dormant cells was up to four times thicker than in regular cells. Once in the environment, A. baumannii is able to survive regardless of the availability of dissolved oxygen, which represents a serious public health concern.

Ultrastructural, Confocal and Viscoelastic Characteristics of Whole Blood and Plasma After Exposure to Cadmium and Chromium Alone and in Combination: An Ex Vivo Study.

BACKGROUND/AIMS: Heavy metal pollution is increasing in the environment, contaminating water, food and air supplies. This can be linked to many anthropogenic activities. Heavy metals are absorbed through the skin, inhalation and/or orally. Irrespective of the manner of heavy metal entry in the body, the blood circulatory system is potentially the first to be affected following exposure and adverse effects on blood coagulation can lead to associated thrombotic disease. Although the plasma levels and the effects of cadmium (Cd) and chromium (Cr) on erythrocytes and lymphocytes have been described, the environmental exposure to heavy metals are not limited to a single metal and often involves metal mixtures, with each metal having different rates of absorption, different cellular, tissue, and organ targets. Therefore the aim of this study is to investigate the effects of the heavy metals Cd and Cr alone and whether Cr synergistically increases the effect of Cd on physiological important processes such as blood coagulation. METHODS: Human blood was exposed to the heavy metals ex vivo, and thereafter morphological analysis was performed with scanning electron- and confocal laser scanning microscopy (CLSM) in conjunction with thromboelastography®. RESULTS: The erythrocytes, platelets and fibrin networks presented with ultrastructural changes, including varied erythrocytes morphologies, activated platelets and significantly thicker fibrin fibres in the metal-exposed groups. CLSM analysis revealed the presence of phosphatidylserine on the outer surface of the membranes of the spherocytic erythrocytes exposed to Cd and Cr alone and in combination. The viscoelastic analysis revealed only a trend that indicates that clots that will form after heavy metal exposure, will likely be fragile and unstable especially for Cd and Cr in combination. CONCLUSION: This study identified the blood as an important target system of Cd and Cr toxicity.