Factor XIII-A Val34Leu and Tyr204Phe variants influence clot kinetics in a cohort of South African type 2 diabetes mellitus patients.

Factor XIII, a transglutaminase that plays a crucial role in clot formation, consists of subunits A and B. Single nucleotide polymorphisms in Factor XIII-A have been linked to thrombotic risk. In Type 2 Diabetes mellitus (T2DM), a hypercoagulable state is thought to contribute to the high mortality rate associated with thrombotic diseases. Due to the lack of prevalence data of FXIII-A single nucleotide polymorphisms (SNPs) in T2DM in a South African cohort, this study assessed the prevalence FXIII-A Val34Leu (rs5985) and Tyr204Phe (rs3024477) SNP's and the effect on clot kinetics in T2DM. MATERIALS AND METHODS: A cohort of T2DM patients (n = 100) and race, age and gender matched healthy controls (n = 101) were recruited following ethical approval. Thromboelastography® (TEG®) was used to assess the viscoelastic properties in platelet poor plasma (PPP) in controls (n = 91) and T2DM patients (n = 91) younger than 50 years old. Genomic DNA was isolated from whole blood using the Quick-DNA™ Miniprep Plus Kit and PCR-RFLP was used to genotype each sample for FXIII-A rs5985 and rs3024477 SNPs. RESULTS: TEG® analyses indicated a longer R-time (p < 0.0001) and higher TMRTG (p < 0.0001) in PPP of T2DM patients. Control and T2DM genotype distribution conformed to Hardy-Weinberg equilibrium (p > 0.05). There was a higher prevalence of the wildtype genotype of FXIII-A Tyr204Phe (rs3024477) SNP in T2DM (OR = 0.23, 95% CI = 0.12-0.42, p < 0.0001). The 204Phe variant was more frequent in the Caucasians (OR = 0.39, 95% CI = 0.05-0.33, p < 0.0001). The presence of the 204Phe variant in T2DM affected TMRTG (p = 0.0207). The variant affected R time (p = 0.0432) and TMRTG (p = 0.0209 and p = 0.0207) in controls and T2DM, respectively. CONCLUSION: An inverse association with T2DM and FXIII-A Tyr204Phe was found. A hypo coagulable PPP clot profile was observed in T2DM. A shorter reaction time was observed and but faster rate at which the clot reached maximum strength in both controls and T2DM in the presence of the 204Phe variant.

Molecular regulation of autophagy in a pro-inflammatory tumour microenvironment: New insight into the role of serum amyloid A.

Chronic inflammation, systemic or local, plays a vital role in tumour progression and metastasis. Dysregulation of key physiological processes such as autophagy elicit unfavourable immune responses to induce chronic inflammation. Cytokines, growth factors and acute phase proteins present in the tumour microenvironment regulate inflammatory responses and alter crosstalk between various signalling pathways involved in the progression of cancer. Serum amyloid A (SAA) is a key acute phase protein secreted by the liver during the acute phase response (APR) following infection or injury. However, cancer and cancer-associated cells produce SAA, which when present in high levels in the tumour microenvironment contributes to cancer initiation, progression and metastasis. SAA can activate several signalling pathways such as the PI3K and MAPK pathways, which are also known modulators of the intracellular degradation process, autophagy. Autophagy can be regarded as having a double edged sword effect in cancer. Its dysregulation can induce malignant transformation through metabolic stress which manifests as oxidative stress, endoplasmic reticulum (ER) stress and DNA damage. On the other hand, autophagy can promote cancer survival during metabolic stress, hypoxia and senescence. Autophagy has been utilised to promote the efficiency of chemotherapeutic agents and can either be inhibited or induced to improve treatment outcomes. This review aims to address the known mechanisms that regulate autophagy as well as illustrating the role of SAA in modulating these pathways and its clinical implications for cancer therapy.

Erythrocyte deformability and eryptosis during inflammation, and impaired blood rheology.

OBJECTIVE: This review focusses on the erythrocytes (RBCs) and their structural changes during inflammation and impaired blood rheology. We discuss systemic inflammation and the effects of dysregulated inflammatory molecules. These pro-inflammatory molecules directly affect the haematological system, and particularly the RBCs, platelets and plasma proteins. We focus on the three main changes; increased RBC eryptosis (programmed cell death, similar to apoptosis) and pathological deformability, platelet hyperreactivity and anomalous blood clotting, due to pathological changes to fibrin(ogen) protein structure. This pro-inflammatory haematological system directly affects blood rheology. In turn, hemorheological parameters such as RBC deformability are important parameters in hypercoagulation, which is a hallmark of inflammation. For RBC deformation to happen during blood flow, the RBC membrane needs to be elastic to elongate sufficiently to squeeze through small capillaries. However, of greater importance is that the cell must return to its original biconcave shape after exiting the small diameter capillaries. CONCLUSION: Hemorheological parameters such as RBC deformability are of great importance clinically, to both identify the presence and extent of inflammation, and to study these parameters during intervention therapies. RBC rheology and deformability may therefore be a useful cell model for pharmaceutical testing.

The stabilizing effect of an oligomeric proanthocyanidin on red blood cell membrane structure of poorly controlled Type II diabetes.

Type II diabetes (T2D) is a pandemic characterized by pathological circulating inflammatory markers, high-glucose levels and oxidative stress. The hematological system is especially vulnerable to these aberrant circulating molecules, and erythrocytes (RBCs) show aberrant rheology properties, owing to the direct contact with these molecules. Pathological levels of circulating inflammatory markers in T2D therefore have a direct effect on the molecular and cellular structure of RBCs. Previous research has suggested that antioxidants may reduce oxidative stress that results from the pathological inflammatory markers. Particularly, polyphenol antioxidants like oligomeric proanthocyanidins (OPCs) may act as a hydroxyl mopping agent, and may have a positive effect on the deformability and membrane protein structure of RBCs from T2D. In this paper, we look at the effect of one such agent, Pinus massoniana bark extract (standardized to 95% oligomeric proanthicyanidins), on the RBC membrane structures and RBC shape changes of T2D, after laboratory exposure at physiological levels. Our methods of choice were atomic force microscopy and scanning electron microscopy to study RBC elasticity and ultrastructure. Results showed that in our hands, this OPC could change both the eryptotic nature of the RBCs, as viewed with scanning electron microscopy, as well as the elasticity. We found a significant difference in variation between the elasticity measurement values between the RBCs before and after OPC exposure (P-value <0.0001). In conclusion, the data from both these techniques therefore suggest that OPC usage might contribute to the improvement of RBC functioning.

Oral v. pulse intravenous cyclophosphamide: A retrospective analysis of adverse events in a setting with a high burden of infectious disease.

BACKGROUND: Cyclophosphamide (CPM) is still considered to be the first-line treatment for many life-threatening autoimmune conditions. It does, however, carry a significant risk of serious adverse events, especially infections. At present CPM is administered as either a daily oral dose (DOC) or an intravenous pulse (PIVC). There is uncertainty regarding the safety profiles of both regimens in settings with a high burden of infectious diseases. OBJECTIVE: To compare the frequency and nature of adverse events related to the use of DOC and PIVC in such a setting. METHODS: A cohort of patients treated with CPM for autoimmune diseases at Tygerberg Academic Hospital, Cape Town, South Africa, from 1 January 2008 to 31 May 2013 was studied. We compared participants receiving DOC and PIVC with regard to disease characteristics and the occurrence of major adverse events. RESULTS: A total of 134 participants (92 DOC and 42 PIVC) were included. Participants in the DOC group were treated for longer (174 v. 101 days; p<0.01) and with higher cumulative doses (17 276 v. 3 327 mg; p<0.01). Risk of infection was similar in the two groups, although there were 6 deaths from leucopenic sepsis in the DOC group (v. 0; p=0.18). Nadir leucocyte counts were also lower in the DOC group (median 3.8 v. 5.3 × 109/L; p=0.02). CONCLUSION: Infection rates in the two groups were similar, but DOC was associated with longer treatment duration, greater cumulative CPM doses and more severe leucopenia. If resources allow and available literature provides support for efficacy, consideration should be given to greater use of PIVC.

Deleterious mutation in the FYB gene is associated with congenital autosomal recessive small-platelet thrombocytopenia.

BACKGROUND: The FYB gene encodes adhesion and degranulation-promoting adaptor protein (ADAP), a hematopoietic-specific protein involved in platelet activation, cell motility and proliferation, and integrin-mediated cell adhesion. No ADAP-related diseases have been described in humans, but ADAP-deficient mice have mild thrombocytopenia and increased rebleeding from tail wounds. PATIENTS AND METHODS: We studied a previously reported family of five children from two consanguineous sibships of Arab Christian descent affected with a novel autosomal recessive bleeding disorder with small-platelet thrombocytopenia. Homozygosity mapping and exome sequencing were used to identify the genetic lesion causing the disease phenotype on chromosome 5. Bone-marrow morphology and platelet function were analyzed. Platelets were characterized by scanning electron microscopy. RESULTS: We identified a homozygous deleterious nonsense mutation, c.393G>A, in FYB. A reduced percentage of mature megakaryocytes was found in the bone marrow. Patients' platelets showed increased basal expression of P-selectin and PAC-1, and reduced increments of activation markers after stimulation with ADP, as detected by flow cytometry; they also showed reduced pseudopodium formation and the presence of trapped platelets between the fibrin fibers after thrombin addition, as observed on scanning electron microscopy. CONCLUSIONS: This is the first report of a disease caused by an FYB defect in humans, manifested by remarkable small-platelet thrombocytopenia and a significant bleeding tendency. The described phenotype shows ADAP to be important for normal platelet production, morphologic changes, and function. It is suggested that mutation analysis of this gene be included in the diagnosis of inherited thrombocytopenia.

Obesity, hypertension and hypercholesterolemia as risk factors for atherosclerosis leading to ischemic events.

Atherosclerosis is a widespread disease of the arterial system that is generated by injury to the vasculature due to hypercholesterolemia, hypertension and inflammatory diseases. In the current review, we discuss the role of different risk factors, including obesity, hypertension and hypercholesterolemia in atherosclerosis, which may ultimately lead to either cardiovascular or cerebral complication. Inflammation plays a pivotal role in conjunction with obesity, hypertension and hypercholesterolemia in the etiology of atherosclerosis. We discuss the role of inflammation with regards to reactive oxygen species (ROS) linked to the specific risk factors. The role of nitric oxide (NO) in conjunction with ROS is also important. Correlations of inflammatory cytokines and their functions in the mentioned risk factors are also discussed. The risk factors may ultimately lead to ischemic events, including transient ischemic attacks (TIAs), thrombotic stroke and myocardial infarction. Importantly, it seems as if there is a combination of pathophysiological triggers that may eventually result in atherosclerosis. Therefore, atherosclerosis is not the result of only one risk factor, but a combination of various physiological processes such as homeostasis and the inflammatory response. Ultimately, each patient's risk profile is unique and determines their immediate risk for acute thrombotic events or lethal ischemia.

Differences in morphology of fibrin clots induced with thrombin and ferric ions and its pathophysiological consequences.

The activation of blood coagulation leads to the formation of thrombin that, in turn, converts soluble plasma fibrinogen into insoluble fibrin clot. In healthy individuals, fibrin is effectively degraded; however, in prothrombotic states, proteolysis of fibrin clots are often delayed or even inhibited, and is associated with altered fibrin structure. We have previously shown that in inflammatory conditions like stroke and diabetes, this fibrin forms dense matted deposits. Although there are several factors that modify fibrin structure and delay fibrinolysis in these conditions, no mechanism is yet known to be responsible for a persistent presence of thrombi in the coronary and/or cerebral circulations. It seems, therefore, desirable to better understand this phenomenon in order to improve the effectiveness of thrombolytic therapies. Here, we show that ferric ions can activate non-enzymatic blood coagulation resulting in the formation of fibrin-like dense matted deposits (DMD) demonstrable by electron scanning microscopy (SEM). These DMDs are similar to those found in stroke and diabetes. On the basis of these findings we can conclude that the spontaneous formation of fibrin-like dense deposits in patients' blood may be a consequence of what is known as iron overload. Therefore, it is possible that inactivation of unbound iron in blood by small molecular weight chelating agents may prevent thrombotic consequences of the excessive accumulation of iron in the circulation.

Hydroxyl radical-modified fibrinogen as a marker of thrombosis: the role of iron.

Excessive free iron in blood and in organ tissues (so called iron overload) has been observed in degenerative diseases such as atherosclerosis, cancer, neurological, and certain autoimmune diseases, in which fibrin-like deposits are also found. Although most of the body iron is bound to hemoglobin and myoglobin in a divalent ferrous form, a certain amount of iron exists in blood as a trivalent (ferric) ion. This particular chemical state of iron has been shown to be toxic to the human body when not controlled by endogenous and/or dietary chelating agents. Experiments described in this paper show for the first time that ferric ions (Fe(3+)) can generate hydroxyl radicals without participation of any redox agent, thus making it a special case of the Fenton reaction. Ferric chloride was also demonstrated to induce aggregation of purified fibrinogen at the same molar concentrations that were used for the generation of hydroxyl radicals. Iron-aggregated fibrinogen, by contrast to native molecule, could not be dissociated into polypeptide subunit chains as shown in a polyacrylamide gel electrophoresis. The mechanism of this phenomenon is very likely based on hydroxyl radical-induced modification of fibrinogen tertiary structure with the formation of insoluble aggregates resistant to enzymatic and chemical degradations. Soluble modified fibrinogen species can be determined in blood of thrombotic patients by the reaction with protamine sulfate and/or by scanning electron microscopy. In view of these findings, it is postulated that iron-induced alterations in fibrinogen structure is involved in pathogenesis of certain degenerative diseases associated with iron overload and persistent thrombosis. It is concluded that the detection of hydroxyl radical-modified fibrinogen may be utilized as a marker of a thrombotic condition in human subjects.

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.

Scanning electron microscopy analysis of erythrocytes in thromboembolic ischemic stroke.

INTRODUCTION: Erythrocytes play an important role in hemostasis and disease conditions. During ischemic stroke, erythrocytes undergo oxidative and proteolytic changes resulting in a changed cellular rheology. METHODS: Blood samples were obtained from controls and thromboembolic ischemic stroke patients (within 48 h of stroke). The ultrastructure of erythrocytes was compared, using a scanning electron microscope (SEM). Abnormal morphology included codocytes, knizocytes, stomatocytes, and echinocytes. Percentage of abnormal cells was calculated, and the analyses were performed using the statistical program NCSS with the level of significance set at 0.05. A t-test was carried out to compare the data from the erythrocyte counts of stroke patients with that of the control subjects. RESULTS: Ultrastructural SEM results showed that there are a large percentage of erythrocytes in healthy individuals that do not have a typical discoid shape, when studying the cells using a high magnification electron microscope. Furthermore, analysis showed that variation in shape is so subtle that it is not clearly visible using a typical light microscopy blood smear analysis. Thromboembolic ischemic stroke patients presented with a significant amount of erythrocytes with abnormal morphology. CONCLUSION: We suggest that in healthy individuals, a typical smear would contain several nondiscoid-shaped erythrocytes, only clearly visible at high magnification. However, thromboembolic ischemic stroke does significantly impact erythorcyte shape, and this change in morphology may result in an impaired microcirculation, as well as impaired oxygen carrying capacity. This changed morphology may further complicate the restoring of homeostasis caused by acute thromboembolic stroke.

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.

The forgotten science: reviving morphology.

Are modern science and clinicians forgetting or ignoring the importance of morphology and microscopy in studying disease and disease patterns? Here we ponder that current science research over-emphasizes the value of molecules and disease modelling, or rather under-estimate the usefulness of microscopy and morphology. We debate the usefulness of morphology in contemporary research and wonder whether our techniques are too old-fashioned or whether our field is seen as redundant.

Histological assessment of SJL/J mice treated with the antioxidants coenzyme Q10 and resveratrol.

The muscular dystrophies (MDs) are genetic disorders of muscle degeneration due to mutations in genes that encode a wide variety of proteins. Dysferlinopathy are characterized by the absence of dysferlin in skeletal muscle and an autosomal recessive mode of inheritance. Both histological and ultrastructural pathology have been well established in dysferlinopathy patients and dysferlin-deficient animal models. To our knowledge the effect of antioxidant supplementation on this level has not been described previously. This article therefore focuses on the histopathology to reveal the effect of antioxidant supplementation. The study aimed to determine, at cellular level, the histopathological changes in the SJL/J mouse model following a 90 day trial with antioxidant supplementation. Markedly reduced inflammatory insult in the more affected quadriceps muscles of animals treated with high doses of CoQ10 and a combination of resveratrol/CoQ10 were observed. The outcome provides evidence that high doses of antioxidant supplementation resulted in decreased dystrophic markers and enhanced tissue integrity at cellular level.

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 geometric morphometric study into the sexual dimorphism of the human scapula.

Sex determination is vital when attempting to establish identity from skeletal remains. Two approaches to sex determination exists: morphological and metrical. The aim of this paper was to use geometric morphometrics to study the shape of the scapula and its sexual dimorphism. The sample comprised 45 adult black male and 45 adult black female scapulae of known sex. The scapulae were photographed and 21 homologous landmarks were plotted to use for geometric morphometric analysis with the 'tps' series of programs, as well as the IMP package. Consensus thin-plate splines and vector plots for males and females were compared. The CVA and TwoGroup analyses indicated that significant differences exist between males and females. The lateral and medial borders of females are straighter while the supraspinous fossa is more convexly curved than that of males. More than 91% of the females and 95% of the males were correctly assigned. Hotelling's T(2)-test yielded a significant p-value of 0.00039. In addition, 100 equidistant landmarks representing the curve only were also assigned. These, however, yielded considerably poorer results. It is concluded that it is better to use homologous landmarks rather than curve data only, as it is most probable that the shape of the outline relative to the fixed homologous points on the scapula is sexually dimorphic.

Influence of acceleration voltage on scanning electron microscopy of human blood platelets.

Scanning electron microscopy (SEM) is used to view a variety of surface structures, molecules, or nanoparticles of different materials, ranging from metals, dental and medical instruments, and chemistry (e.g. polymer analysis) to biological material. Traditionally, the operating conditions of the SEM are very important in the material sciences, particularly the acceleration voltage. However, in biological sciences, it is not typically seen as an important parameter. Acceleration voltage allows electrons to penetrate the sample; thus, the higher the acceleration voltage the more penetration into the sample will occur. As a result, ultrastructural information from deeper layers will interfere with the actual surface morphology that is seen. Therefore, ultimately, if acceleration voltage is lower, a better quality of the surface molecules and structures will be produced. However, in biological sciences, this is an area that is not well-documented. Typically, acceleration voltages of between 5 and 20 kV are used. This manuscript investigates the influence of acceleration voltages ranging from 5 kV to as low as 300 V, by studying surface ultrastructure of a human platelet aggregate. It is concluded that, especially at higher magnifications, much more surface detail is visible in biological samples when using an acceleration voltage between 2 kV and 300 V.

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.

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.