Adverse effects of copper, manganese and mercury, alone and in mixtures on the aorta and heart of Spraque-Dawley rats.

Cardiovascular diseases (CVD) are a common global cause of death and are therefore a major health concern. Inhaled or ingested environmental heavy metals contribute to the development of CVD. The aim of this study was to address the limited information available on the effect of relevant dosages of metals in mixtures. Three metals with reported effects on the cardiovascular system (CVS) were identified, and these metals were copper (Cu), manganese (Mn) and mercury (Hg). In Sprague-Dawley rats, the adverse effects of copper (Cu), manganese (Mn) and mercury (Hg), alone and as part of mixtures, on the blood parameters, the aorta and heart were investigated. Forty-eight male Sprague-Dawley rats were randomly divided into eight groups (n = 6): control, Cu, Mn, Hg, Cu + Mn, Cu + Hg, Mn + Hg and Cu, Mn + Hg. The seven experimental groups received the metal mixtures at 100 times the World Health Organisation (WHO) safety limit for drinking water (2 mg/L for Cu, 0.4 mg/L for Mn and 0.06 mg/L for Hg) via oral gavage for 28 days. After 28 days, compared with the control, red blood cell levels were increased for Cu + Hg. All other measured blood parameters were unchanged. Morphological changes in the tunica media were connective tissue deposition and an abundance of collagen type I in the metal exposed aortic tissues. In the cardiac tissue of metal-exposed rats, changes in the cardiomyocyte and myofibrillar arrangement, with an increase in collagen type I and III was observed. Ultrastructurally, the aortic collagen and elastin band arrangement and the cardiac mitochondrial and myofibrillar arrangement and structures were altered in the experimental groups. These changes indicated that exposure to these metals in rats caused minor changes in the blood parameters, however, the changes in tissue and cellular structure indicated an increased risk for the development of CVD.

Investigating the ultrastructural and viscoelastic characteristics of whole blood after exposure to the heavy metals cadmium, lead and chromium, alone and in combination.

In recent years, heavy metal exposure has become a serious health concern as more humans are being exposed to heavy metals on a daily basis. Most of the environmental contamination and human exposure result from anthropogenic activities such as mining and smelting. The industrial and agricultural sectors also play a big role. Cigarette smoke in particular contains trace amounts of heavy metals that put chronic smokers at serious risk. Previous studies have determined that there is a strong correlation between heavy metal exposure and adverse effects on the coagulation system. The aim of this study was to investigate the effect of cadmium, lead and chromium alone and in combination on erythrocytes and fibrin networks that form part of the coagulation system as well as the viscoelastic properties of thrombus formation by using thromboelastography®. The choice of metals for this study was based on a previous study that compared the levels of metals between smokers and nonsmokers and found significantly higher levels of cadmium, lead and chromium in the platelet-rich fibrin of smoking individuals. Scanning electron microscopy analysis revealed that the cadmium and chromium combination groups caused the highest degree of echinocyte formation and fibrin network alterations. These findings were supported by the thromboelastography® analysis that indicated a significant decrease in reaction-time and split point values for the chromium-containing group, suggesting a shorter initiation time for clot formation. The findings of this study support the hypothesis that the coagulation pathway is a potential target for heavy metal toxicity.

Whole blood ultrastructural alterations by mercury, nickel and manganese alone and in combination: An ex vivo investigation.

The distribution of metals across the environment is increasingly becoming a major concern as they not only pollute the environment but also pose a danger to humans and animals. Human exposure to heavy metals often occurs as a combination of metals the synergistic effects of which can be more toxic than a single metal. The aim of this study was to investigate the effects that the metals mercury (Hg), nickel (Ni) and manganese (Mn) alone and in combination have on erythrocyte morphology and other components of the coagulation system using the haemolysis assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy. Human blood was exposed to the heavy metals ex vivo, and percentage haemolysis was determined. Ultrastructural analysis of erythrocytes, platelets and fibrin networks was performed using SEM. Analysis of phosphatidylserine (PS) flip-flop was determined using confocal laser scanning microscopy. At the highest concentration of 10,000× the World Health Organization safety limit, all the metals caused haemolysis. The results showed that the exposure of erythrocytes to Hg alone and in combination with other metals displayed more haemolysis compared to Ni and Mn alone and in combination. Components of the coagulation system showed ultrastructural changes, including the formation of echinocytes and the activation of platelets with all single metals as well as the combinations. Confocal laser scanning microscopy analysis showed the presence of PS on the outer surface of the echinocytes that were exposed to metals alone and in combination. It can, therefore, be concluded that these heavy metals have a negative impact on erythrocytes and the coagulation system.

Investigating the effect of a platelet additive solution on apheresis platelet and fibrin network ultrastructure.

In thrombotic events and diseases such as cancer, HIV/AIDS, dysfibrinogenaemia, as well as acute incidents (e.g. burn wounds), ultrastructure of platelets and fibrin networks change. In the current study, we compare the ultrastructure of platelets and fibrin networks of apheresis platelets stored in citrated human plasma (CP) and in a first-generation platelet additive solution (PAS) (T-Sol), to that of fresh donor plasma (FP). Eighteen apheresis platelet donors donated platelets on Trima®-Accel™ V5.2 and V5.1 cell separators. Six collections were stored for five days in autologous citrated plasma (CP); six collections were stored in 40% citrated human plasma and 60% PAS solution (CP/PAS) controlled, for the duration of storage, at a constant temperature (22±2°C) with continuous flat-bed agitation; and six collections were stored in conditions uncontrolled for temperature and without continuous agitation. On days 1, 3 and 5, equal volumes of human thrombin were mixed with platelets collected in either CP or CP/PAS to form a coagulum (fibrin network containing platelet aggregates), followed by preparation for scanning electron microscopy. Results were compared with platelets and fibrin networks in FP. Typically, in FP, platelet aggregates with smooth membranes and pseudopodia are seen and fibrin networks arrange to form major, thick fibers and scattered, minor, thin fibers. On day 1, in CP and in all CP/PAS units, platelet ultrastructure compared well to that of FP, although the fibrin fibers were denser, with the minor fibers forming a matted layer over the major fibers. On day 3, in platelet units uncontrolled for temperature and without continuous agitation during storage, some platelet aggregates in CP/PAS showed typical apoptotic morphology, with shrinkage and membrane damage, but comparable fibrin networks were present. On day 5 however, in those units where storage conditions were uncontrolled and where the pH had decreased to below 6.4, no platelet aggregates were seen and fibrin was arranged into short, lumpy masses with no separate major or minor fibrin fibers visible. In those units stored at 22°C with continuous flat-bed agitation, where pH was maintained >7.0, ultrastructure of platelets and fibrin network in CP/PAS was typical and similar to FP and CP at the end of five days of storage. Examining platelet and fibrin network ultrastructure may be useful, in addition to conventional laboratory analysis, in assessing the viability and potential clinical efficacy of platelets for transfusion and could play a role in the evaluation of new generation platelet additive solutions.

A review of immunomodulators with reference to Canova.

Immunomodulators are substances which modify the immunity of an individual to favour a particular immunological response. The immune response and the function of the immune response regulation process are described, with special reference to cancer and autoimmune disease. Homeopathy and its role in immune regulation are discussed with special reference to Canova. Canova is a homeopathic product produced, according to the Hahnemannian homeopathic method, in Brazil. Its role in cancer, bone marrow and haematopoiesis as well as macrophage and monocyte activation is reviewed. Canova seems to stabilize platelet morphology in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). The data suggest that the future of immunomodulators and homeopathic products which appear to have an effect on the immune response requires a better understanding of the relative need for immune activation versus immune modulation. Homeopathic products specifically need more attention.

Oxidative and haemostatic effects of copper, manganese and mercury, alone and in combination at physiologically relevant levels: An ex vivo study.

Water contamination with metals due to anthropogenic activity is increasing and subsequent exposure increases the risk of associated toxicity. Exposure is not limited to a single metal but usually involves mixtures of different metals at different concentrations. Little is known about the contribution of this type of exposure, in humans, to the development of non-communicable diseases such as cardiovascular disease, and an increased risk to thrombosis. The World Health Organization has established limits for metal levels in drinking water and this includes levels for copper (Cu), manganese (Mn) and mercury (Hg). In this study, at 100X these limits, the ability of the metals' oxidative effects as catalysts of the Fenton reaction and/or ability to bind glutathione (GSH) were determined. The haemostatic effects of these metals, alone and in combination, at the World Health Organization limit were then evaluated. The ultrastructural and viscoelastic alterations of exposed ex vivo whole blood were also evaluated using scanning electron microscopy and thromboelastography® (TEG), respectively. Cu, alone and in combination with Mn and/or Hg, induced hydroxyl radical formation and reduced GSH levels. Ex vivo exposure caused deformation of erythrocytes and accelerated platelet activation especially for Cu, alone and in combination, with Mn. Reduction in the lysis potential of the clot was also observed for all combinations, especially Cu in combination with Hg as well as Mn alone. Although the TEG findings were not statistically significant, the trends indicate that the exposure to these metals, alone and in combination, adversely affects thrombus formation in ex vivo blood, thereby potentially increasing the risk in exposed individuals for thrombosis.

Ultrastructure of activated mouse platelets: a qualitative scanning electron microscopy study.

Platelets form an integral part of the coagulation process, and their ultrastructure can provide valuable information regarding diseases associated with hemostasis. During coagulation, platelets aggregate; this aggregation can be achieved in vitro, by adding thrombin to platelet-rich plasma. Previous research showed that human thrombin could be used successfully to activate mouse platelets. When conservative changes are included, the amino acid similarity between human and mouse thrombin is approximately 75%. In this qualitative study, we compare the ultrastructure of mouse platelet aggregates activated by human thrombin as well as two concentrations of mouse thrombin, using the scanning electron microscope. Results show that both human and mouse thrombin activate platelets to form aggregates with typical pseudopodia formation. Magnification up to 250,000x showed membrane morphology with the open canalicular system pores visible in both the mouse- and human-activated platelets. It is therefore concluded that mouse platelets can be successfully aggregated using either mouse or human thrombin.

Evaluation of the morphological changes in the lungs of BALB/c mice after inhalation of spherical and rod-shaped titanium nanoparticles.

Titanium nanoparticles are widely used by industry in consumer products such as sunscreens and some cosmetic products due to their specifically engineered properties. Some of these properties may, however, increase the toxicity of the nanoparticles which in turn may affect human and environmental health. Therefore, it is of utmost importance to study the possible effects of these particles through in vivo studies, which might produce different results than in vitro cell studies. The current study aimed to investigate the possible remodelling in the lungs of BALB/c mice by means of light and transmission electron microscopy after inhalation of spherical and rod-shaped titanium nanoparticles at two different concentrations. The focus of this paper was to demonstrate whether whole body exposure to different concentrations of the said nanoparticles could induce an inflammatory response in the lungs and no inter particle comparison was done or retention investigated. Animals were divided into five experimental groups: control, high and low concentration groups exposed to the spherical-shaped particles, as well as high and low concentration groups exposed to the rod-shaped particles. Histological and ultrastructural changes, typical of an inflammatory response, were noted in the lungs of the exposed animals. These changes were not observed in the lungs of the control animals. It can be concluded from this study that titanium nanoparticles may cause inflammatory reactions in the lungs of animals exposed through inhalation, as indicated by the presence of inflammatory cells and congestion of inter-alveolar areas. This has implications for individuals who may be potentially exposed during the production and use of titanium nanoparticles.

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies.

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the 'natural state' fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.

Investigating lung remodeling in Modul8-treated BALB/c asthmatic animals.

Animal models of bronchial hyperresponsiveness have been successfully used to investigate the pathophysiology of asthma. When mice are sensitized and challenged with an allergen, such as OVA, they experience symptoms and processes similar to that of humans, and are therefore widely used as asthmatic animal models. In the current study the BALB/c murine asthmatic animal model was used to investigate the histological and ultrastructural changes that occur in the lungs of asthmatic animals that received no treatment, compared to two groups of asthmatic animals that were treated with a homeopathic immunodulator Modul8 and hydrocortisone as positive control, respectively. Eosinophil counts in the bronchial lavage of the animals were also analyzed, since it is known that eosinophil counts are increased in the bronchial lavage in asthma. Results indicated that eosinophil counts were elevated in asthmatic animals compared to the controls, but were found to be significantly decreased in the treatment groups. Also, in the asthmatic, untreated animals, histological and ultrastructural changes, typically associated with the inflammatory process were found. Both treatment groups compared well to that of the control animals, indicating that the homeopathic product might be successfully used in the treatment of asthma.

Investigating the ultrastructure of platelets of HIV patients treated with the immuno-regulator, Canova: a qualitative scanning electron microscopy study.

The resistance of HIV strains to the available antiretroviral medication has become a major problem in the world today. This has forced researchers to investigate the possible use of alternative drugs such as homeopathic medicine (e.g. immunomodulators) to enhance the immune system of patients infected with HIV. Canova is an immunomodulator of herbal origin which is known to stimulate the host defense against several pathological states through the activation of the immune system. Blood platelets play an important role in homeostasis, thrombosis and the immune response by forming platelet aggregates. The ultrastructure of platelet aggregates of patients with HIV has been studied previously using SEM to determine the effect of HIV on the platelet morphology. Membrane blebbing and ruptured platelet membranes were observed which is indicative of apoptosis, revealing that HIV patients may develop thrombocytopenia as a result of peripheral platelet destruction. The aim of the current study was to investigate the effect of HIV on the morphology of platelets from patients treated with the immuno-modulator, Canova, compared to control individuals and HIV patients not on the Canova treatment. Blood was drawn from the individuals and the coagula were formed by adding human thrombin to the platelet rich plasma. Examination was done using SEM. CD4 counts were also determined. Slight morphological changes were seen when comparing the fibrin networks from the control, untreated HIV patients and the Canova-treated HIV patients, suggesting that HIV does not impact on the fragility of fibrin networks. In HIV patients there are bleb-like bulges on the membrane of platelets as well as membrane breakages visible on the aggregate, whereas in the Canova-treated patients membrane blebbing is far less pronounced and there are large areas of intact, smooth membranes with visible canalicular areas, suggesting that Canova protects the membranes of platelets and that blebbing does not appear in such great proportions as was found in the untreated HIV group. These results support and provide ultrastructural evidence for the results seen in previous research, where it is seen that Canova protects the immune system of immuno-compromised patients by keeping the ultrastructure intact thereby preventing the devastating cyto-destructive effects of HIV disease.

Comparative scanning electron microscopy of platelets and fibrin networks of human and differents animals / Microscopía electrónica de barrido comparativa de plaquetas y redes de fibrina de humano y de diferentes animales

The purpose of the present study was to compare the platelet and fibrin network ultrastructure of humans to eight different animal species in order to determine the differences between human and animal platelet and fibrin morphology, and to determine whether the animals studied differ in their platelet and fibrin morphology, and whether these differences can be observed by scanning electron microscopy. Platelets and fibrin networks play an important role both in the coagulation process as well as physiologically in allergic processes and immunological mechanisms. The thickness of human fibrin networks were compared to mouse (Mus musculus), equine (Equus caballus), vervet monkey (Chlorocebus aethiops previously Cercopithecus aethiops), oryx (Oryx gazella), ovine (Ovis aries), penguin (Spheniscus demersus), rabbit (Oryctolagus cuniculus) and sea turtle (Caretta caretta). Fibers were measured and divided into thin (minor) fibers, intermediate fibers and thick (major) fibers. The results obtained indicated that for each of the three fibrin classes, the size ranges of the monkey, oryx and equine were not significantly different to one another, and the human, penguin, oryx and ovine not significantly different to one other. From these results it can be concluded that mammals and aves possess a distinct tri-modal fibrin fiber distribution, different from that of the studied reptilian species where the sea turtle possesses a distinct bimodal fibrin fiber distribution and it can be suggested that the utilization of mammalian and avian models, in terms of fibrin fiber distribution patterns, might be a suitable alternative for ultrastructural studies.

El propósito del presente estudio fue comparar la ultraestructura de plaquetas y las redes de fibrina de los seres humanos y de ocho diferentes especies de animales, con el fin de determinar las diferencias morfológicas de estas estructuras y si las diferencias pueden ser observadas por microscopía electrónica de barrido. Las plaquetas y las redes de fibrina desempeñan un papel importante tanto en el proceso de coagulación como, fisiológicamente en procesos alérgicos y mecanismos inmunológicos. Elgrosor de las redes de fibrina humana fue comparado con las del ratón (Mus musculus), equino (Equus caballus), mono vervet (Chlorocebus aethiops, anteriormente Cercopithecus aethiops, antílope Africano (Oryx gazella), ovino (Ovis aries), pingüino (Spheniscus demersus), conejo (Oryctolagus cuniculus) y tortuga marina (Caretta caretta). Las fibras fueron medidas y agrupadas en fibras delgadas (menor), fibras intermedias y fibras gruesas (grandes). Los resultados obtenidos indicaron que para cada una de las tres clases de fibrina, los rangos de su tamaño en el mono, antílope africano y en equino no fueron significativamente diferentes entre sí, mientras que en humano, pingüino, antílope africano y ovino no fueron significativamente diferentes entre éstos. De estos resultados se pudo concluir que mamíferos y aves poseen una distribución tri-modal de fibras de fibrina, distinta a la de las especies de reptiles estudiadas, donde la tortuga de mar posee una distribución bimodal de fibras de fibrina. Se puede sugerir que la utilización de los modelos mamíferos y aviar, en términos de patrones de distribución de fibras de fibrina, pueden ser una alternativa adecuada para los estudios ultraestructurales.