Toe-walking and its impact on first and second rocker in gait patterns with different degrees of artificially emulated soleus and gastrocnemius contracture.

BACKGROUND: Toe-walking is one of the most common gait deviations (due to soleus and/or gastrocnemius muscle contractures), compromising the first (heel rocker) and second (ankle rocker) of the foot during walking. The aim of this study is to evaluate the effect of emulated artificially gastrocnemius and soleus contractures on the first and second rocker during walking. METHOD: An exoskeleton was built to emulate contractures of the bilateral gastrocnemius and soleus muscles. Ten healthy participants were recruited to walk under the following conditions: without emulated contractures or with bilateral emulated contractures at 0°,10°, 20° and 30° of plantarflexion of the soleus or gastrocnemius in order to create an artificial restriction of dorsiflexion ankle movement. A linear regression from the ankle plantar-dorsiflexion angle pattern was performed on 0-5 % of the gait cycle (first rocker) and on 12-31 % of the gait cycle (second rocker) to compute the slope of the curve. The proportion of participants with the presence of the first and second rocker was then computed. A Statistical Parametric Mapping (SPM) analysis assessed the kinematic variations among different degrees of emulated contractures. FINDINGS: The first and second rockers are completely absent from 10° of plantarflexion emulated contracture. The data indicate there was a non-linear shift of the gait pattern of the ankle kinematics and an important shift toward plantarflexion values with the loss of the rockers. INTERPRETATION: This study suggests that toe-walking in the experimental simulation situation is not necessarily due to a high emulated contracture level and can occur with a small emulated contracture by an adaptation choice. This study may improve interpretation of clinical gait analysis and shows that the link between the level of gastrocnemius/soleus emulated contracture and progression of toe-walking (increased plantarflexion during gait) is not linear.

S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas.

Despite marked success in treatment with immune checkpoint inhibitor (CPI), only a third of patients are responsive. Thus, melanoma still has one of the highest prevalence and mortality rates; which has led to a search for novel combination therapies that might complement CPI. Aberrant methylomes are one of the mechanisms of resistance to CPI therapy. S-adenosylmethionine (SAM), methyl donor of important epigenetic processes, has significant anti-cancer effects in several malignancies; however, SAM's effect has never been extensively investigated in melanoma. We demonstrate that SAM modulates phenotype switching of melanoma cells and directs the cells towards differentiation indicated by increased melanogenesis (melanin and melanosome synthesis), melanocyte-like morphology, elevated Mitf and Mitf activators' expression, increased antigen expression, reduced proliferation, and reduced stemness genes' expression. Consistently, providing SAM orally, reduced tumor growth and progression, and metastasis of syngeneic BRAF mutant and wild-type (WT) melanoma mouse models. Of note, SAM and anti-PD-1 antibody combination treatment had enhanced anti-cancer efficacy compared to monotherapies, showed significant reduction in tumor growth and progression, and increased survival. Furthermore, SAM and anti-PD-1 antibody combination triggered significantly higher immune cell infiltration, higher CD8+ T cells infiltration and effector functions, and polyfunctionality of CD8+ T cells in YUMMER1.7 tumors. Therefore, SAM combined with CPI provides a novel therapeutic strategy against BRAF mutant and WT melanomas and provides potential to be translated into clinic.

Mechanisms of human FoxP3+ Treg cell development and function in health and disease.

Regulatory T (Treg ) cells represent an essential component of peripheral tolerance. Given their potently immunosuppressive functions that is orchestrated by the lineage-defining transcription factor forkhead box protein 3 (FoxP3), clinical modulation of these cells in autoimmunity and cancer is a promising therapeutic target. However, recent evidence in mice and humans indicates that Treg cells represent a phenotypically and functionally heterogeneic population. Indeed, both suppressive and non-suppressive Treg cells exist in human blood that are otherwise indistinguishable from one another using classical Treg cell markers such as CD25 and FoxP3. Moreover, murine Treg cells display a degree of plasticity through which they acquire the trafficking pathways needed to home to tissues containing target effector T (Teff ) cells. However, this plasticity can also result in Treg cell lineage instability and acquisition of proinflammatory Teff cell functions. Consequently, these dysfunctional CD4+ FoxP3+ T cells in human and mouse may fail to maintain peripheral tolerance and instead support immunopathology. The mechanisms driving human Treg cell dysfunction are largely undefined, and obscured by the scarcity of reliable immunophenotypical markers and the disregard paid to Treg cell antigen-specificity in functional assays. Here, we review the mechanisms controlling the stability of the FoxP3+ Treg cell lineage phenotype. Particular attention will be paid to the developmental and functional heterogeneity of human Treg cells, and how abrogating these mechanisms can lead to lineage instability and Treg cell dysfunction in diseases like immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, type 1 diabetes, rheumatoid arthritis and cancer.

Kinematics can help to discriminate the implication of iliopsoas, hamstring and gastrocnemius contractures to a knee flexion gait pattern.

BACKGROUND: Excessive Knee Flexion Gait Pattern (KFGP) is a common gait deviation in many pathological conditions. The contractures of the muscles that have been identified as being responsible of KFGP are: iliopsoas, hamstring and gastrocnemius. RESEARCH QUESTION: How do isolated contractures of the iliopsoas, hamstrings and gastrocnemius impact knee flexion during gait? METHODS: Three levels of contracture (mild, moderate and severe) were simulated bilaterally using an exoskeleton on 10 healthy participants for iliopsoas, hamstring and gastrocnemius muscles. A gait analysis session was performed to evaluate the joint kinematics according to the different simulated contractures. Thirty one parameters were chosen to analyze the kinematics of the thorax, pelvis, hip, knee and ankle. A principal component analysis (PCA) was used to determine the kinematic parameters influenced by contractures. RESULTS: In addition to a permanent knee flexion observed for the three muscles with contracture: the contracture of the iliopsoas induces a large hip flexion with pronounced anterior pelvis tilt; the contracture of the hamstrings induces an ankle dorsiflexion during the support phase with a posterior pelvis tilt; the contracture of the gastrocnemius induces an absence of first and second rocker of the ankle with a slight flexion of hip and a slight anterior pelvis tilt. SIGNIFICANCE: These results support the identification of the muscles responsible for a KFGP. A better knowledge of the interactions between contractures and associated joint kinematics of the same and adjacent joints will support the interpretation of gait analyses by more precisely and faster targeting the concerned muscle.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

Influence of different degrees of bilateral emulated contractures at the triceps surae on gait kinematics: The difference between gastrocnemius and soleus.

INTRODUCTION: Ankle plantarflexion contracture results from a permanent shortening of the muscle-tendon complex. It often leads to gait alterations. The objective of this study was to compare the kinematic adaptations of different degrees of contractures and between isolated bilateral gastrocnemius and soleus emulated contractures using an exoskeleton. METHODS: Eight combinations of contractures were emulated bilaterally on 10 asymptomatic participants using an exoskeleton that was able to emulate different degrees of contracture of gastrocnemius (biarticular muscle) and soleus (monoarticular muscle), corresponding at 0°, 10°, 20°, and 30° ankle plantarflexion contracture (knee-flexed and knee-extended). Range of motion was limited by ropes attached for soleus on heel and below the knee and for gastrocnemius on heel and above the knee. A gait analysis session was performed to evaluate the effect of these different emulated contractures on the Gait Profile Score, walking speed and gait kinematics. RESULTS: Gastrocnemius and soleus contractures influence gait kinematics, with an increase of the Gait Profile Score. Significant differences were found in the kinematics of the ankles, knees and hips. Contractures of soleus cause a more important decrease in the range of motion at the ankle than the same degree of gastrocnemius contractures. Gastrocnemius contractures cause greater knee flexion (during the stance phase) and hip flexion (during all the gait cycle) than the same level of soleus contractures. CONCLUSION: These results can support the interpretation of the Clinical Gait Analysis data by providing a better understanding of the effect of isolate contracture of soleus and gastrocnemius on gait kinematics.

Feasibility and reliability of using an exoskeleton to emulate muscle contractures during walking.

Contracture is a permanent shortening of the muscle-tendon-ligament complex that limits joint mobility. Contracture is involved in many diseases (cerebral palsy, stroke, etc.) and can impair walking and other activities of daily living. The purpose of this study was to quantify the reliability of an exoskeleton designed to emulate lower limb muscle contractures unilaterally and bilaterally during walking. An exoskeleton was built according to the following design criteria: adjustable to different morphologies; respect of the principal lines of muscular actions; placement of reflective markers on anatomical landmarks; and the ability to replicate the contractures of eight muscles of the lower limb unilaterally and bilaterally (psoas, rectus femoris, hamstring, hip adductors, gastrocnemius, soleus, tibialis posterior, and peroneus). Sixteen combinations of contractures were emulated on the unilateral and bilateral muscles of nine healthy participants. Two sessions of gait analysis were performed at weekly intervals to assess the reliability of the emulated contractures. Discrete variables were extracted from the kinematics to analyse the reliability. The exoskeleton did not affect normal walking when contractures were not emulated. Kinematic reliability varied from poor to excellent depending on the targeted muscle. Reliability was good for the bilateral and unilateral gastrocnemius, soleus, and tibialis posterior as well as the bilateral hamstring and unilateral hip adductors. The exoskeleton can be used to replicate contracture on healthy participants. The exoskeleton will allow us to differentiate primary and compensatory effects of muscle contractures on gait kinematics.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo.

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii.

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.

Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa.

The production of secondary metabolites in seaweed have been related to a capability to partition compounds into cellular specialized storage structures, like gland cells and the corps en cerise (CC) or cherry bodies. The possible mechanisms that bring these compounds to the thallus surface remain poorly understood. Therefore, the aim of this work is perform a characterization of the CC and determine the intra-cellular dynamics of halogenated compounds in Laurencia obtusa. The dynamics of CC and the mechanisms related to the intra-cellular transport of halogenated compounds were evaluated by using optical tweezers and time-lapse video microscopy. The CC were isolated and its elemental composition was characterized using X-ray microanalysis. The cellular distribution of halogenated compounds was also demonstrated by fluorescence microscopy. Three-dimensional reconstruction technique was used to provide a visualization of the structures that connect CC to cell periphery. As main findings, we confirmed that the halogenated compounds are mainly found in CC and also in vesicles distributed along the cytoplasm and within the chloroplasts. We demonstrated that CC is mechanically fixed to cell periphery by a stalk-like connection. A vesicle transport though membranous tubular connections was seen occurring from CC to cell wall region. We also demonstrated a process of cortical cell death event, resulting in degradation of CC. We suggested that the vesicle transportation along membranous tubular connections and cell death events are related to the mechanisms of halogenated compounds exudation to the thallus surface and consequently with defensive role against herbivores and fouling.

Ultrastructure of the Miracidium of Echinostoma paraensei Lie and Basch, 1967 (Trematoda, Echinostomatidae).

The morphology of Echinostoma paraensei was studied using transmission electron microscopy. The terebratorium region has many electrondense secretory granules and many folds on the surface. The epidermal cells that cover the larval body have unique nuclear morphology, many mitochondria and vesicles being attached to the interepidermal ridges by a septate junction. The cilia present the organization 9 + 2 and a typical structure with a shaft, axosome, basal body and rootlet. Below the epidermal cells there is a layer of circular muscle and, adjacent to it, a layer of longitudinal muscle fibers. The excretory system has two flame cells, with internal and external ribs and leptotriches at the barrel region, an excretory vesicle and an excretory pore.

Aspects of the early moments of interaction between tachyzoites of Toxoplasma gondii with neutrophils.

The interaction of the tachyzoite stage of Toxoplasma gondii with neutrophils was investigated. Morphological aspects of the initial moments of interaction were analyzed by transmission and scanning electron microscopy, revealing at least three types of reaction from the leukocytes to the parasite: the projection of filopodia, formation of a tunnel-like projection involving the parasite, and invagination of the leukocyte surface at the point of entry. The influence on infectivity of tyrosine kinase, phosphokinase C and phosphatidylinositol 3 kinase cell signaling pathways were studied with the aid of drugs affecting these enzymes in these cells.

Morphology of the rediae of Echinostoma paraensei (Trematoda: Echinostomatidae) from its intermediate host Lymnaea columella (Mollusca, Gastropoda).

The morphology of the rediae of Echinostoma paraensei obtained from Lymnaea columella was studied using light, scanning and transmission electron microscopy. The measurements of the mature rediae differ from those described originally, and the taxonomic importance of the ambulatory buds and papilliform process is discussed. Uniciliated papillae were observed in the mouth region. The birth papilla is a bulb-like structure, well defined at the anterior end of the body of the rediae, which opens through a split. There are no microvilli in the tegument surface of the larvae, but numerous tegumental folds, varying according to the contraction of the body of the rediae. The outer syncytial layer is located on a thick basal lamina below which the circular and the longitudinal muscle fiber layers are located.

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles.

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.

Platelet-activating factor modulates a secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum.

In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum.

Veneza zonata (Hemiptera: Coreidae)/trypanosomatid relationship: action of hemolymph in vitro and experimental infection.

The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.

Antileishmanial activity of an indole alkaloid from Peschiera australis.

In this study, we show the leishmanicidal effects of a chloroform fraction (CLF) and a purified indole alkaloid obtained from crude stem extract of Peschiera australis against Leishmania amazonensis, a causative agent of cutaneous leishmaniasis in the New World. In a bioassay-guided chemical fractionation, the leishmanicidal activity in CLF completely and irreversibly inhibited promastigote growth. This fraction was also active against amastigotes in infected murine macrophages. Chemical analysis of CLF identified an iboga-type indole alkaloid coronaridine as one of its major compounds. Coronaridine showed potent antileishmanial activity, inhibiting promastigote and amastigote growth. Promastigotes and amastigotes treated with CLF or coronaridine showed pronounced alterations in their mitochondria as assessed by transmission electron microscopy.

Megasome biogenesis in Leishmania amazonensis: a morphometric and cytochemical study.

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process.

The social climate in chronic haemodialysis units as perceived by patients and nurses.

The purpose of this research was to check if there was a correlation between how patients perceived the atmosphere in dialysis units compared to the nurses' perception. The intention was to examine commonality and differences between the two groups. This would serve as a basis to change the behaviour of the nurses and/or to develop or create settings that are more suitable. Sixteen haemodialysis units in Israel participated in this study, a total of 190 participants of which ninety-three were nurses and ninety-seven were patients. Patients and nurses gave their consent in writing and answered anonymously. The Moos Ward Atmosphere Scale was used using 100 true and false questions. These questions were condensed into six main categories. Our results showed that the patients and staff had significantly different perceptions in the following categories: 1. Openness and sensitivity, 2. Staff attitude, 3. Order and organization, 4. Mutual support, 5. New treatment approaches. The greatest degree of agreement between the two groups was found in only one category: that of the "doctor's attitude" or behaviour. Three studies have been found that have investigated the unit atmosphere as perceived by patients and staff-two in Israel in an oncology and a psychiatric unit. Rhodes did the third study in a haemodialysis unit in the Unites States and his results are compared with the results in this study. The differences found between nurses and patients show that there is a communication problem. It is recommended that interpersonal communication be improved to close the gap in perceptions, thereby improving the unit atmosphere. New strategies should be developed for coping and helping the patient to adjust.

Morphological changes during conoid extrusion in Toxoplasma gondii tachyzoites treated with calcium ionophore.

Treatment of tachyzoites of Toxoplasma gondii with the calcium ionophore A23187 induced dramatic ultrastructural changes that were observed by light and electron microscopy. Light microscopy showed a higher percentage (22%) of tachyzoites with the conoid extruded when compared to control parasites. Electron microscopy confirmed the conoid extrusion by both transmission and scanning electron microscopy. Freeze-fracture replicas showed that the plasma membrane adjacent to cytoplasmic dense granules appeared devoid of intramembranous particles. Membrane-limited vesicles and filopodium-like structures at the cell surface were observed in treated cells. 3-D reconstruction from serial sections confirmed the data and showed a heterogeneity in dense granule shape not reported in control cells.