TROP-2 expression in papillary thyroid cancer: a preliminary cyto-histological study.

OBJECTIVE: TROP-2 (human trophoblast cell surface marker) is a gene-related protein expressed in trophoblastic cells, which is also present in a variety of epithelial cancers and whose overexpression has been found to correlate with a poor prognosis. We analysed the possibility of using the expression of TROP-2 to detect papillary thyroid carcinoma (PTC) on cytological and histological samples, and compared it with Hector Battifora mesothelial antigen-1 (HBME-1). METHODS: From 127 patients, 127 fine needle aspirates (FNAs), in which HBME-1 was detected by immunocytochemistry (ICC), were re-classified according to the Bethesda system for reporting thyroid cytopathology (TBSRTC): 20% were benign, 56% were atypical cells/follicular lesion of undetermined significance (AUS/FLUS), 4% were follicular neoplasm/suspicious for a follicular neoplasm, 5% were suspicious for malignancy and 16% were malignant. Sufficient material to test for TROP-2 was available in 64 FNAs, 22 of which had a histological control. Including six additional cases in which the FNAs were not available, immunohistochemistry (IHC) was carried out with both markers on 94 cases. RESULTS: Among 88 FNAs with histological control, the sensitivity of HBME-1 to predict PTC was 87.5% (28/32) and the specificity was 86% (48/56), whereas, in 22 FNAs, TROP-2 sensitivity was 100% (13/13) and specificity was 89% (8/9). In 94 histological specimens in which IHC was carried out with both markers, the sensitivity and specificity were 82% and 86%, respectively, for HBME-1 and 87% and 89%, respectively, for TROP-2. The difference between the markers was not significant. Concordance between IHC and ICC was 76% for HBME-1 and 91% for TROP-2. CONCLUSION: TROP-2 can be used as well as HBME-1 in thyroid cytology to detect PTC. Positivity for either or both markers could help to stratify the risk of malignancy in indeterminate FNAs. Larger studies are need to analyse its role in the behaviour of PTC and its variants.

MicroRNA expression profiling in male and female familial breast cancer.

BACKGROUND: Gender-associated epigenetic alterations are poorly investigated in male and female familial breast cancer (fBC). MicroRNAs may contribute to the different biology in men and women particularly related to RASSF1A pathways. METHODS: Microarray technology was used to evaluate miRNA profile in 24 male and 43 female fBC. Key results were validated using RT-qPCR in an external samples set. In vitro studies were carried out to verify microRNA-target gene interaction. RESULTS: Pathway enrichment analysis with the 287 differentially expressed microRNAs revealed several signalling pathways differently regulated in male and female cases. Because we previously hypothesised a peculiar involvement of RASSF1A in male fBC pathogenesis, we focussed on the MAPK and the Hippo signalling pathways that are regulated by RASSF1A. Male miR-152 and miR-497 upregulation and RASSF1A and NORE1A interacting gene downregulation were observed, confirming a possible indirect interaction between miRNAs and the two genes. CONCLUSIONS: For the first time, a different microRNA expression pattern in male and female fBC has been shown. Moreover, the importance of RASSF1A pathway in male fBC carcinogenesis has been confirmed, highlighting a possible role for miR-152 and miR-497 in controlling MAPK and Hippo signalling pathways, regulated by RASSF1A.

New and old microbial communities colonizing a seventeenth-century wooden church.

The North of Romania is known for its wooden churches dating from the seventeenth and eighteenth centuries. Their deterioration constitutes a major problem due to their value for the cultural heritage. The microbial community from a seventeenth-century wooden church (Nicula, Romania) was investigated by characterization of uncultivated and cultivated bacteria using 16S rDNA sequence analysis. The study revealed not only the prevalence of the Bacillus thuringiensis strain IAM 12077 but also the presence of new microbial communities of Planomicrobium and Variovorax that were not previously reported in paintings or on wood. The identification of fungi showed the presence of seven common genera found on the walls and icon surfaces. Common bacteria from the human oral microbiota were not identified in the bacterial community.

In vitro evaluation of the effects of yttria-alumina-silica microspheres on human keratinocyte cells.

The behavior of yttria-alumina-silica spray-dried microspheres was investigated in vitro on a human keratinocyte cell line, first to exclude their cytotoxicity. The HaCaT cells were chosen due to their well-characterized phenotype and their phagocytic ability. Microscopic analysis and cell viability tests showed no negative effect of the microspheres on cells morphology and behavior. Scanning electron microscopy and transmission electron microscopy results evidenced the cellular internalization of the microspheres at 48 h after their incubation with cultured cells. The shape, size distribution, structure, composition, and chemical states of the elements on samples surface were analyzed by SEM, transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy, because these properties could influence their internalization by cells. The yttrium distribution on the microspheres surface was indicated by fluorescence microscopy imaging. The microspheres dimension and shape inside the cells was in accordance with their dimension and shape before incubation. The microspheres seemed captured and engulfed by the cells in native form and appeared resistant to degradation over the first 48 h. Most of the analyzed cells took up more microspheres, suggesting that the microspheres were actively phagocytosed by the cells and accumulated within the cytoplasm. X-ray photoelectron spectroscopy results on Al and Si atomic environments denoted Al-O-Si crosslinks, which improve the surface protection to corrosion.

Interface processes between iron containing aluminosilicate systems and simulated body fluid enriched with protein.

The behaviour of iron containing aluminosilicate samples in Kokubo's simulated body fluid (SBF) and in SBF enriched with bovine serum albumin (BSA) has been investigated. Crystalline samples of (80-x)SiO2 X 20Al2O3 X Fe2O3 system, with x = 5, 10 or 15 mol%, obtained by sol-gel method and heat treated at 1200 degrees C in air for 24 h. Data on electrical conductivity, calcium, phosphorous and potassium concentrations in simulated body fluids after samples soaking in static regime at 37 degrees C, for several periods up to 14 days, were used to estimate the dynamics of these cations on the interface of aluminosilicate samples with SBF, and with SBF containing BSA. The UV-visible and fluorescence spectra recorded from the simulated body fluids after immersion of the investigated aluminosilicate samples evidence changes function on immersion time and Fe2O3 content.

[Endophthalmitis or TASS--case report]. / Endoftalmita sau TASS--caz clinic.

We present the case of a 65-years-old female with a history of endophthalmitis after residual cortex aspiration postextracapsular cataract extraction. The patient was admitted for cataract surgery on the other eye. After phacoemulsification she developed a series of signs and symptoms on the anterior eye segment which plead for the diagnosis of endophthalmitis or toxic anterior segment syndrome (TASS). Under specific therapy the evolution has been favorable.

[The genetic polymorphism of MTHFR gene in schizophrenia]. / Polimorfismul genetic al genei MTHFR in schizofrenie.

UNLABELLED: The 677C > T polymorphism of the MTHFR gene, resulting in hyperhomocysteinemia, has been shown to be implicated in the aetiology of schizophrenia. Previous studies showed that A1298 C polymorphism seems not to be related to schizophrenia. AIM OF THE STUDY: To analyze two genetic polymorphisms of the MTHFR gene, 677C > T and A1298 C in 44 patients with schizophrenia and evaluate its relationship with the risk of schizophrenia and with some clinical aspects. MATERIAL AND METHOD: We determined the presence of the 677C > T and A1298 C mutations of the MTHFR gene in 44 inpatients with schizophrenia and in 35 normal controls. The patients were assessed by psychiatric examination and scalar evaluation. RESULTS: 28 (66,7%) of the patient group had the T allele of the 677C > T genetic polymorphism, compared to 11 (34,3%) subjects of the control group. The intensity of the positive, negative and general symptoms was slightly higher in the patients presenting the T allele. The A1298C missense mutation was more frequent between control subjects (57,5%) compared to the patient group (39%). The intensity of the positive symptoms was slightly increased in the patients with the missense mutation in the position 1298, but the intensity of the negative and general symptoms did not differ. CONCLUSIONS: Our study confirms the role of the 677C > T genetic polymorphism in the susceptibility for schizophrenia. The relationship between A1298C genetic polymorphism and schizophrenia was not demonstrated in our study.

Association of bovine papillomavirus type-2 and urinary bladder tumours in cattle from Romania.

In cattle, bracken fern toxicity is characterized by the presence of haematuria and tumours of the urinary bladder of both epithelial and mesenchymal origin. This syndrome is known as chronic enzootic hematuria (CEH) and is also present in Romania. From January 2006 to April 2007, 90 urinary bladders from slaughtered cows originating from hill-mountain area of Neamt county (Romania), where CEH is endemic, were collected. All samples were histologically examined and Bovine papillomavirus type 2 (BPV-2) DNA was detected by polymerase chain reaction (PCR) analysis in 68% of the analyzed tumours samples. BPV-2 positive urinary bladder tumours were also immunohistochemically analysed for the expression of the major viral oncoprotein E5. We found the expression of E5 intracytoplasmically with a typical juxtanuclear pattern. E5 expression was not observed in normal mucosa, suggesting a causal role for this protein in the neoplastic process. This is the first report of BPV-2 infection in Eastern European country, confirming the role of BPV-2 in naturally occurring bovine urothelial carcinogenesis.

Quantitative and qualitative approach of glycan-glycan interactions in marine sponges.

Cell recognition and adhesion involving many kinds of cell surface molecules operate via homotypic and/or heterotypic protein-protein and protein-carbohydrate binding. Our investigations in marine sponges have provided direct evidence for a novel molecular mechanism of multivalent glycan-glycan binding related to cellular interactions. Biochemical characterization of purified proteoglycans revealed the presence of specific acidic glycans, different from classical glycosaminoglycans. Such acidic glycans of high molecular weight, containing fucose, glucuronic or galacturonic acids, and pyruvate and sulfate groups may represent a new class of primordial proteoglycans, named by us glyconectins. The thermodynamic and kinetic approaches of biological macromolecule interactions do not provide a direct measurement of the intermolecular binding forces that are fundamental for the function of the ligand-receptor association. Using the atomic force microscopy (AFM), we provided the first quantitative evaluation of the binding strength between cell adhesion proteoglycans. Measurement of binding forces intrinsic to cell adhesion glyconectin proteoglycans (AGPs) is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. (i) As a model, we selected the cell AGP isolated from the marine sponge Microciona prolifera; it mediates in vivo cell recognition and aggregation via homotypic, species-specific, multivalent, and calcium ion-dependent glycan-glycan interactions. (ii) Under physiological conditions, a large cohesive force theoretically able to hold the weight of approximately 1600 cells was measured. (iii) The C-2 autocomplementarity model for AGP-AGP interactions; and (iv) the requirement of the calcium ionic bridges suggest also that the self-recognition and multivalency of glycan-glycan interactions are essential for cell adhesion. (v) The evolution of glyconectin-like proteoglycan molecules may have been a fundamental prerequisite for the emergence of the first multicellular organisms. Glycan-glycan interactions may thus provide a new paradigm for molecular self-recognition.

Disruption of NF-kappa B signaling and chemokine gene activation by retroviral mediated expression of IKK gamma/NEMO mutants.

Phosphorylation of I kappa Bs--the cytoplasmic inhibitors of the NF-kappa B transcription factors--is the key event which triggers activation of the NF-kappa B cascade. Signal-mediated phosphorylation of I kappa B alpha is mediated by a multiprotein complex, the I kappa B kinase (IKK) complex, which is composed of at least three identified subunits. Two of these polypeptides, IKK alpha and IKK beta, also known as IKK1 and IKK2, are the catalytic subunits of the kinase complex and phosphorylate I kappa B alpha and I kappa B beta. The third component, NEMO/IKK gamma, does not exhibit kinase activity, but rather constitutes a regulatory subunit. In the present study, C-terminal truncated forms of IKK gamma--Delta C-IKK gamma 306 and Delta C-IKK gamma 261--were stably expressed in the myeloid cell line U937 by retroviral-mediated gene transfer. Overexpression of Delta C-IKK gamma resulted in a reduction in IKK kinase activity in vitro, a subsequent decrease in NF-kappa B DNA binding activity, and inhibition of chemokine gene induction in response to TNFalpha stimulation or paramyxovirus infection. This study demonstrates the efficacy of Delta C-IKK gamma as a repressor of IKK signaling and NF-kappa B activation and suggests a potential gene therapy approach to limit chronic inflammation due to chemokine hyperactivation.

Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii.

The precise molecular mechanisms underlying the switch between the two developmental stages of Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly understood. Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism. We have previously described a cDNA encoding a bradyzoite-specific enolase, ENO1. We now report the isolation and characterization of another enolase-encoding cDNA (ENO2) that is expressed preferentially in the tachyzoite stage. The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity. They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK). We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities. T. gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in Escherichia coli. While ENO1 and ENO2 display similar K(m) values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at V(max) compared with that of the bradyzoite-specific enolase (ENO1). Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct. Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism.

Detection of stenoses in the anterior circulation using frequency-based transcranial color-coded sonography.

Atherosclerotic stenoses of the intracranial vessels are less frequent than those of the extracranial vessels, but they are associated with a considerable annual stroke rate. The aim of the present study was to investigate the usefulness of frequency-based transcranial color-coded sonography (TCCS), transcranial Doppler sonography (TCD) and digital subtraction angiography (DSA) in patients with middle cerebral artery (MCA) and intracranial internal carotid artery (ICA) stenosis. Forty patients presenting with 48 intracranial stenoses of the anterior circulation were involved in the study. The stenoses were detected in the neurovascular laboratory during routine TCD examinations. All patients underwent an additional frequency-based TCCS examination. Both the axial and coronal planes were obtained to allow the exact localization of MCA stenosis and differentiation from intracranial ICA stenosis. Angle-corrected flow velocity measurements were performed if straight vessel compartments were 20 mm or more in length. A total of 18 stenoses (44%) were investigated additionally with DSA. According to the investigation with TCD, 20 (42%) stenoses were low-grade, 12 (25%) were moderate, and the remaining 16 (33%) were severe. Angle-corrected flow velocity measurements obtained with the integrated pulse-wave Doppler device of the TCCS machine were highly correlated (0.912, p < 0.001) with those obtained with TCD. TCCS achieved a reliable differentiation of MCA main stem stenosis vs. intracranial ICA stenosis in 7 patients and vs. MCA branch stenosis in 4 patients, but TCD failed in these two subgroups. The agreement between DSA and TCCS to evaluate semiquantitatively 18 intracranial stenoses resulted in a weighted-kappa value of 0.764. The major clinically relevant advantages of TCCS over TCD in MCA stenosis are its ability to differentiate MCA trunk stenosis from terminal ICA or MCA branch stenosis reliably and to perform angle-corrected flow velocity measurements.

The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes. Implications for evolutionary origin of apicomplexans.

The recent discovery of a vestigial, nonphotosynthetic plastid ("apicoplast") in the Apicomplexa has considerably modified our perception of the evolutionary origin of these parasites. Phylogenetic analysis and the presence of four surrounding membranes of the apicoplast provide important support for the hypothesis that apicomplexans have acquired their apicoplast by secondary endosymbiosis, probably from a green alga. This suggests that genes encoding predicted homologs of proteins of green algae or related photosynthetic lineages could have entered the nucleus of apicomplexan parasites by transfer from the ancestor harboring the apicoplast. We describe here complementary DNAs encoding two Toxoplasma gondii glycolytic enzymes, glucose-6-phosphate isomerase (G6-PI) and enolase, which have considerable identities with land plant counterparts. Both cDNAs of T. gondii complement Escherichia coli mutants lacking G6-PI and enolase genes and lead to the expression of active enzymes. In the drug untreatable encysted bradyzoites of T. gondii, G6-PI and enolase genes are overexpressed or exclusively expressed at both transcriptional and protein levels. Moreover, three-dimensional models and protein phylogeny confirmed that G6-PIs and enolases of T. gondii, Plasmodium falciparum, and land plants are closely related. Because these glycolytic enzymes are plant homologs, which differ from those of animals, they will be useful to trace the evolutionary origin of Apicomplexa and might offer novel chemotherapeutic targets in diseases caused by apicomplexan parasites.

Serum lipid picture of rabbits fed on silicate-supplemented atherogenic diet.

Silicon is recognised as a protective trace element in atherosclerosis by epidemiologic and biochemical studies. The goal of the present work was the evaluation of the antiatheromatous effect of the natrium silicate in an experimental trial on 30 rabbits given a standard diet (control), an atherogenic diet (L1 lot) and a natrium silicate-supplemented atherogenic diet (L2 lot), by comparing the variation of the lipid pictures between each of the experimental lots and control. Levels of total lipids, cholesterol, triglycerides (TG), free fatty acids (FFA) and phospholipids indicated that the natrium silicate supplemented to the atherogenic diet minimised the lipid metabolism unbalance by keeping constant the level of FFA and TG in rabbits.

Strontium-induced repetitive calcium spikes in a unicellular green alga

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

A new 1-MHz probe for transcranial Doppler sonography in patients with inadequate temporal bone windows.

Recent large-scale transcranial Doppler sonography (TCD) studies have revealed that, in 3%-5% of patients, the temporal bone window is insufficient for examination with 2-MHz probes. The diagnostic value of a new 1-MHz probe for TCD was compared with a standard 2-MHz probe in patients with an insufficient ultrasonic window. From a total population of 514 consecutive patients, the study involved only patients in whom a bad or absent temporal bone window was revealed during 2-MHz TCD examination. Fifty patients (39 women and 11 men) with a mean age of 71 +/- 9 y were investigated with a 2-MHz probe and subsequently with a 1-MHz probe. Using the 2-MHz probe, 18 patients (group I) lacked a temporal insonation window bilaterally, while 12 had an absent bone window unilaterally (group II). Twenty patients (group III) had bilateral bone windows, but with insufficient detection of the anterior and posterior cerebral arteries. In four of the 18 patients in group I (22%), the circle of Willis could be demonstrated with the 1-MHz probe. In 11 of the 12 patients in group II (92%), it was possible to demonstrate the contralateral vessels through the ipsilateral bone window with the 1-MHz probe, while this was only possible in 25% with the 2-MHz probe. All 20 patients of group III could be successfully examined with the 1-MHz probe. The 1-MHz probe provides an opportunity to perform TCD studies in older patients presenting with absent or insufficient temporal bone windows for examination with 2-MHz probes.

Emboli detection during continuous-wave doppler sonography of internal carotid artery stenosis.

BACKGROUND AND PURPOSE: Continuous-wave (cw)-Doppler sonography is a wellestablished noninvasive method for examining the extracranial vessels. It is extremely rare for an acute ischemic event to occur during cw-Doppler sonography of carotid stenosis. However, no data are available concerning the influence of mechanical stress connected with this procedure on the emboli rate distal to the stenosis. METHODS: The study involved 46 patients (28 men, 18 women) aged 66+/-10 years with an unilateral moderate (n=10) or severe (n=36) stenosis of the internal carotid artery (ICA). Twenty patients had a symptomatic stenosis and the remaining 26 were asymptomatic. Patients with other embolic sources, such as atrial fibrillation or mechanical heart valves, were excluded. All patients underwent bilateral emboli detection of the middle cerebral artery for a period of 29+/-8 minutes. In a second step, emboli detection (5+/-1 minutes) was performed simultaneously with cw-Doppler sonography of the carotid stenosis. RESULTS: Seven of 46 patients (15%) had a median emboli rate of 5 emboli per hour distal to the carotid stenosis before the procedure. During cw-Doppler sonography of the carotid stenosis, 6 patients (13%) had a median emboli rate of 14 emboli per hour without clinical symptoms of cerebral ischemia. One patient had no change of the emboli rate and 2 patients showed a nonsignificant increase in emboli rate during cw-Doppler sonography. Three patients had no emboli before cw-Doppler sonography, but between 12 and 15 emboli per hour during the extracranial examination (P<.001). CONCLUSIONS: It must be presumed that patients with severe stenoses of the ICA represent a subgroup with fragile atheromatous plaques, which are more vulnerable to mechanical stress. The observations support the hypothesis that mechanical stress associated with routine ultrasound examinations of carotid stenosis may, in rare cases, evoke asymptomatic artery-to-artery embolism.

Do chronic middle cerebral artery stenoses represent an embolic focus? A multirange transcranial Doppler study.

BACKGROUND AND PURPOSE: It remains uncertain whether the annual stroke risk of 7% to 8% in middle cerebral artery (MCA) stenosis is of embolic or hemodynamic origin. Preliminary reports provide evidence of emboli exiting from acute MCA stenoses, detected by transcranial Doppler (TCD) sonography. With multirange monitoring before and after the stenosis, TCD monitoring may help for the first time to differentiate microemboli exiting from the MCA stenosis from those with a source proximal to the MCA stenosis. We searched for microembolic signals (MES) using multigated monitoring in patients with chronic MCA stenoses. METHODS: Fifty-eight patients with 78 chronic stenoses of the MCA were enrolled in the study. Additional sources of embolism were ruled out by extensive clinical workup. Twenty-four patients were treated with coumarin, whereas 28 patients received aspirin. The remaining 6 patients discontinued their medication after a few weeks. The sample volume of the multirange probe was placed on either side of the stenotic area of the MCA. RESULTS: Twenty-three (29.5%) of the stenoses were low grade, 18 (23%) were moderate, and 37 (47.5%) were severe. Thirty-seven (47%) of the stenoses were symptomatic and 41 (53%) were asymptomatic before study entry. During follow-up, 2 strokes and 7 transient ischemic attacks occurred. Computer tomography revealed two watershed-type infarcts. Sufficient insonation of the prestenotic and poststenotic segments of the MCA was possible in 70 stenoses (90%). No MES could be detected during a total of 1740 minutes' monitoring time distal to the MCA stenoses, regardless of the patients' medication. MES were also absent in the contralateral MCA. CONCLUSIONS: MES are not detectable in patients with chronic MCA stenoses of different degrees. No MES were found in either symptomatic or asymptomatic stenoses, regardless of the patients' medication. These results indicate that chronic MCA stenoses do not represent a significant embolic source. The absence of MES in the prestenotic Doppler sample volume, the watershed-type infarcts during follow-up, and the absence of small-vessel disease on computed tomography suggests that hemodynamic mechanisms are responsible for recurrent cerebral ischemia.