Prashilla Soma

Changes in red blood cell membrane structure in type 2 diabetes: a scanning electron and atomic force microscopy study

Red blood cells (RBCs) are highly deformable and possess a robust membrane that can withstand shear force. Previous research showed that in diabetic patients, there is a changed RBC ultrastructure, where these cells are elongated and twist around spontaneously formed fibrin fibers. These changes may impact erythrocyte function. Ultrastructural analysis of RBCs in inflammatory and degenerative diseases can no longer be ignored and should form a fundamental research tool in clinical studies. Consequently, we investigated the membrane roughness and ultrastructural changes in type 2 diabetes. Atomic force microscopy (AFM) was used to study membrane roughness and we correlate this with scanning electron microscopy (SEM) to compare results of both the techniques with the RBCs of healthy individuals. We show that the combined AFM and SEM analyses of RBCs give valuable information about the disease status of patients with diabetes. Effectiveness of treatment regimes on the integrity, cell shape and roughness of RBCs may be tracked, as this cell’s health status is crucial to the overall wellness of the diabetic patient.

Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS)

Alzheimer-type dementia (AD) is a neurodegenerative disorder and the most common form of dementia. Patients typically present with neuro- and systemic inflammation and iron dysregulation, associated with oxidative damage that reflects in hypercoagulability. Hypercoagulability is closely associated with increased fibrin(ogen) and in AD patients fibrin(ogen) has been implicated in the development of neuroinflammation and memory deficits. There is still no clear reason precisely why (a) this hypercoagulable state, (b) iron dysregulation and (c) increased fibrin(ogen) could together lead to the loss of neuronal structure and cognitive function. Here we suggest an alternative hypothesis based on previous ultrastructural evidence of the presence of a (dormant) blood microbiome in AD. Furthermore, we argue that bacterial cell wall components, such as the endotoxin lipopolysaccharide (LPS) of Gram-negative strains, might be the cause of the continuing and low-grade inflammation, characteristic of AD. Here, we follow an integrated approach, by studying the viscoelastic and ultrastructural properties of AD plasma and whole blood by using scanning electron microscopy, Thromboelastography (TEG®) and the Global Thrombosis Test (GTT®). Ultrastructural analysis confirmed the presence and close proximity of microbes to erythrocytes. TEG® analysis showed a hypercoagulable state in AD. TEG® results where LPS was added to naive blood showed the same trends as were found with the AD patients, while the GTT® results (where only platelet activity is measured), were not affected by the added LPS, suggesting that LPS does not directly impact platelet function. Our findings reinforce the importance of further investigating the role of LPS in AD.

Poorly controlled type 2 diabetes is accompanied by significant morphological and ultrastructural changes in both erythrocytes and in thrombin-generated fibrin: implications for diagnostics

We have noted in previous work, in a variety of inflammatory diseases, where iron dysregulation occurs, a strong tendency for erythrocytes to lose their normal discoid shape and to adopt a skewed morphology (as judged by their axial ratios in the light microscope and by their ultrastructure in the SEM). Similarly, the polymerization of fibrinogen, as induced in vitro by added thrombin, leads not to the common ‘spaghetti-like’ structures but to dense matted deposits. Type 2 diabetes is a known inflammatory disease. In the present work, we found that the axial ratio of the erythrocytes of poorly controlled (as suggested by increased HbA1c levels) type 2 diabetics was significantly increased, and that their fibrin morphologies were again highly aberrant. As judged by scanning electron microscopy and in the atomic force microscope, these could be reversed, to some degree, by the addition of the iron chelators deferoxamine (DFO) or deferasirox (DFX). As well as their demonstrated diagnostic significance, these morphological indicators may have prognostic value.

Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes.

Diabetes is a condition defined by hyperglycaemia and these patients have a high risk of thrombosis. Previous research showed that ultrastructural changes in clot formation occur in patients in whom there are changes in the coagulation system due to, for example, an inflammatory condition. In the current study, the ultrastructures of platelets and fibrin networks were investigated in 25 diabetic patients. Plasma smears, with and without the addition of thrombin, were prepared. Results indicated that the fibrin network in all diabetic patients had thickened masses of thin, minor fibres over the major fibres, a profile typical of an inflammatory condition. A changed platelet membrane ultrastructure could also be observed in the diabetic patients that revealed typical apoptotic morphology, in whom membrane blebbing could be seen. It can, therefore, be concluded that in diabetic patients, the ultrastructure of fibrin networks show a typical systemic inflammatory profile, although platelets seem to be apoptotic.

Interplay between ultrastructural findings and atherothrombotic complications in type 2 diabetes mellitus

Accelerated atherosclerosis is the main underlying factor contributing to the high risk of atherothrombotic events in patients with diabetes mellitus and atherothrombotic complications are the main cause of mortality. Like with many bodily systems, pathology is observed when the normal processes are exaggerated or uncontrolled. This applies to the processes of coagulation and thrombosis as well. In diabetes, in fact, the balance between prothrombotic and fibrinolytic factors is impaired and thus the scale is tipped towards a prothrombotic and hypofibrinolytic milieu, which in association with the vascular changes accompanying plaque formation and ruptures, increases the prevalence of ischaemic events such as angina and myocardial infarction. Apart from traditional, modifiable risk factors for cardiovascular disease like hypertension, smoking, elevated cholesterol; rheological properties, endogenous fibrinolysis and impaired platelet activity are rapidly gaining significance in the pathogenesis of atherosclerosis especially in diabetic subjects. Blood clot formation represents the last step in the athero-thrombotic process, and the structure of the fibrin network has a role in determining predisposition to cardiovascular disease. It is no surprise that just like platelets and fibrin networks, erythrocytes have been shown to play a role in coagulation as well. This is in striking contrast to their traditional physiological role of oxygen transport. In fact, emerging evidence suggests that erythrocytes enhance functional coagulation properties and platelet aggregation. Among the spectrum of haematological abnormalities in diabetes, erythrocyte aggregation and decreased deformability of erythrocytes predominate. More importantly, they are implicated in the pathogenesis of microvascular complications of diabetes. The morphology of platelets, fibrin networks and erythrocytes are thus essential role players in unravelling the pathogenesis of cardiovascular complications in diabetic subjects.

Smoking and fluidity of erythrocyte membranes: a high resolution scanning electron and atomic force microscopy investigation.

Smoking affects the general health of an individual, however, the red blood cells (RBCs) and their architecture are particularly vulnerable to inhaled toxins related to smoking. Smoking is one of the lifestyle diseases that are responsible for the most deaths worldwide and an individual who smokes is exposed to excessive amounts of oxidants and toxins which generate up to 10(18) free radicals in the human body. Recently, it was reported that smoking decreases RBC membrane fluidity. Here we confirm this and we show changes visible in the topography of RBC membranes, using scanning electron microscopy (SEM). RBC membranes show bubble formation of the phospholipid layer, as well as balloon-like smooth areas; while their general discoid shapes are changed to form pointed extensions. We also investigate membrane roughness using atomic force microscopy (AFM) and these results confirm SEM results. Due to the vast capability of RBCs to be adaptable, their state of well-being is a major indication for the general health status of an individual. We conclude that these changes, using an old technique in a novel application, may provide new insights and new avenues for future improvements in clinical medicine pertaining to conditions like COPD.

An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus.

The study suggests that patients with systemic lupus erythematosus (SLE) present with distinct inflammatory ultrastructural changes such as platelets blebbing, generation of platelet-derived microparticles, spontaneous formation of massive fibrin network and fusion of the erythrocytes membranes. Lupoid platelets actively interact with other inflammatory cells, particularly with white blood cells (WBCs), and the massive fibrin network facilitates such an interaction. It is possible that the concerted actions of platelets, erythrocytes and WBC, caught in the inflammatory fibrin network, predispose to pro-thrombotic states in patients with SLE.

Major involvement of bacterial components in rheumatoid arthritis and its accompanying oxidative stress, systemic inflammation and hypercoagulability

We review the evidence that infectious agents, including those that become dormant within the host, have a major role to play in much of the etiology of rheumatoid arthritis and the inflammation that is its hallmark. This occurs in particular because they can produce cross-reactive (auto-)antigens, as well as potent inflammagens such as lipopolysaccharide that can themselves catalyze further inflammagenesis, including via β-amyloid formation. A series of observables coexist in many chronic, inflammatory diseases as well as rheumatoid arthritis. They include iron dysregulation, hypercoagulability, anomalous morphologies of host erythrocytes, and microparticle formation. Iron dysregulation may be responsible for the periodic regrowth and resuscitation of the dormant bacteria, with concomitant inflammagen production. The present systems biology analysis benefits from the philosophical idea of “coherence,” that reflects the principle that if a series of ostensibly unrelated findings are brought together into a self-consistent narrative, that narrative is thereby strengthened. As such, we provide a coherent and testable narrative for the major involvement of (often dormant) bacteria in rheumatoid arthritis. Impact statement Rheumatoid arthritis (RA) is accompanied by long-term inflammation that is mediated by cytokines and cross-reactive (auto-)antigens. Here we suggest one explanation is the presence of a (dormant) microbiome in RA that sheds the highly potent inflammagen, lipopolysaccharide lipopolysaccharides (LPS) to catalyze inflammagenesis, including via β-amyloid formation. We discuss various co-existing features in RA, including iron dysregulation, hypercoagulability, anomalous morphologies of host erythrocytes, and microparticle formation. We review literature and provide coherent evidence that an aberrant blood microbiome in RA has a major involvement in the development, progression, and therefore over-all etiology of the disease.

Scanning electron microscopy of fibrin networks in rheumatoid arthritis: a qualitative analysis

Rheumatoid arthritis is a chronic inflammatory condition that affects mainly synovial joints and has an impact on approximately 1% of the Western population. The coagulation process is altered in this condition, and this is frequently complicated by thrombocytosis. Changes in fibrin morphology have been linked with inflammation, and this, in turn, plays an important role in thrombosis. Changes in the fibrin fiber formation cause the alterations observed in thrombus morphology. In the current study, the ultrastructure of platelets and fibrin networks was investigated to determine whether any morphological changes are present in these structures in patients suffering from rheumatoid arthritis. Six patients diagnosed with rheumatoid arthritis took part in this study, and their clot morphology was compared to that of control subjects. Citrated blood with and without the addition of thrombin was used. Results indicated that the fibrin networks in the arthritis patients formed thick, matted layers. This matted appearance is due to a changed ultrastructure of the minor, thin fibers. Also, in these patients, spontaneous networks were created without the addition of thrombin, which indicates an abnormal hemostatic protein functioning, and the latter is expressed as visible changes in ultrastructure.

Albumin stabilizes fibrin fiber ultrastructure in low serum albumin type 2 diabetes.

Abstract Serum albumin is an essential plasma protein that serves an important function in maintaining osmotic pressure. Low levels of this protein are associated with the kidney failure and hemodialysis that are often seen in diabetic patients who are at high risk of thrombotic events. In diabetes, fibrin fiber nets are changed to form dense matted deposits (DMDs, or parafibrin). Here the authors investigate whether parafibrin is also present in diagnosed low-albumin diabetes patients and whether the addition of human albumin to plasma from low-albumin diabetes type 2 individuals may change the architecture of the fibrin nets. The authors show that the addition of albumin to plasma of low-albumin diabetes patients progressively caused the DMDs typically found in these patients to revert back to ultrastructure typically seen in healthy individuals. This disease has an extremely complicated pathophysiology and thus cannot be considered as a simple condition. This study shows that serum albumin levels may play an important role in the structure of fibrin fibrils, making them more susceptible to the fibrinolytic degradation and elimination from the circulation.