Szabolcs Felszeghy

Accuracy of In Vivo Carotid B-Mode Ultrasound Compared With Pathological Analysis Intima-Media Thickening, Lumen Diameter, and Cross-Sectional Area

Background and Purpose— This study aimed to determine the correlation of in vivo ultrasound measurements of intima-media thickening (IMT), lumen diameter, and cross-sectional area of the common carotid artery (CCA) with corresponding measurements obtained by gross pathology and histology. Methods— Sixty-six moribund neurological patients (mean age 71 years) underwent B-mode ultrasound of the CCA a few days before death. During autopsy, carotid specimens were removed in toto. Carotid arteries were ligated and cannulated for injection of a hydrophilic embedding material under standardized conditions. The carotid bifurcation was frozen and cut manually in 3-mm cross slices. Digital image analysis was carried out to determine the diameter and the cross-sectional area of the frozen slices of the CCA. IMT was assessed by light microscope. Ultrasonic and planimetric data were compared. Results— Mean measurements of lumen diameter and cross-sectional area were 7.13±1.27 mm and 0.496±0.167 cm2, respectively, by ultrasound, and 7.81±1.45 mm and 0.516±0.194 cm2, respectively, by planimetric analysis of the unfixed redistended carotid arteries (R2=0.389 and 0.497). The mean IMT was 1.005±0.267 mm by ultrasound and 0.67±0.141 mm histologically, resulting in a mean difference of −31%. Conclusions— Transcutaneous B-mode ultrasound provides a reliable approach for in vivo measurements of the cross-sectional area and, less exactly, of the lumen diameter of the CCA. Compared with histological results, in vivo ultrasound measurements of the IMT are systematically larger.

Splitting placodes: effects of bone morphogenetic protein and Activin on the patterning and identity of mouse incisors

SUMMARY The single large rodent incisor in each jaw quadrant is evolutionarily derived from a mammalian ancestor with many small incisors. The embryonic placode giving rise to the mouse incisor is considerably larger than the molar placode, and the question remains whether this large incisor placode is a developmental requisite to make a thick incisor. Here we used in vitro culture system to experiment with the molecular mechanism regulating tooth placode development and how mice have thick incisors. We found that large placodes are prone to disintegration and formation of two to three small incisor placodes. The balance between one large or multiple small placodes was altered through the regulation of bone morphogenetic protein (BMP) and Activin signaling. Exogenous Noggin, which inhibits BMP signaling, or exogenous Activin cause the development of two to three incisors. These incisors were more slender than normal incisors. Additionally, two inhibitor molecules, Sostdc1 and Follistatin, which regulate the effects of BMPs and Activin and have opposite expression patterns, are likely to be involved in the incisor placode regulation in vivo. Furthermore, inhibition of BMPs by recombinant Noggin has been previously suggested to cause a change in the tooth identity from the incisor to the molar. This evidence has been used to support a homeobox code in determining tooth identity. Our work provides an alternative interpretation, where the inhibition of BMP signaling can lead to splitting of the large incisor placode and the formation of partly separate incisors, thereby acquiring molar‐like morphology without a change in tooth identity.

Hyaluronan accumulates around differentiating neurons in spinal cord of chicken embryos.

One major component of the extracellular matrix is hyaluronan (HA) which is thought to play a crucial role in the development of different organs including the central nervous system (CNS). HA is bound by specific receptors, CD44 and RHAMM, depending on cell types of CNS. However, data are lacking on the relation of HA to different cell populations in developing CNS. To provide new data about the co-localization of HA with the various cellular structures of the developing spinal cord, we studied the distribution pattern of hyaluronan in chicken embryos at Hamburger-Hamilton (HH) stages 8-39. A biotinylated HA-binding complex was used in combination with immunohistochemistry for proliferating and differentiating neurons. The intensity of the HA signal was determined by digital densitometry from histological sections. We found three mediolaterally oriented layers in the HA distribution pattern in stage HH23: (1) a moderate HA signal was detected in the ventricular zone; (2) strong HA accumulation was measured around Lim1,2-expressing cells (differentiating neurons) and early MNR2-expressing neurons (early motoneurons), corresponding to the intermediate zone; (3) a strong pericellular HA reaction was found around the neurons of the marginal zone. Interestingly, the peripheral nerves did not show HA signals. These findings suggest a crucial role of HA during neuronal development. We propose that HA may be involved in cell migration and axonal growth in the developing spinal cord.

Retinoids produced by macrophages engulfing apoptotic cells contribute to the appearance of transglutaminase 2 in apoptotic thymocytes.

Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types including T cells. Though the expression of the enzyme was strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 could be detected, when thymocytes were induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the in vivo induction of the enzyme in apoptotic thymocytes. Previous studies have shown that one of these signals is transforming growth factor-β (TGF-β) which is released by macrophages engulfing apoptotic cells. Besides TGF-β, the TG2 promoter contains retinoic acid response elements as well. Here we show that in vitro retinoic acids, or TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes, and the apoptotic signal contributes to the TG2 induction. Inhibition of retinoic acid synthesis either by alcohol or retinaldehyde dehydrogenases significantly attenuates the in vivo induction of TG2 following apoptosis induction indicating that retinoids indeed might contribute in vivo to the apoptosis-related TG2 expression. What is more, the in vivo apoptosis induction in the thymus is accompanied by an enhanced retinoid-dependent transcriptional activity due to the enhanced retinoid synthesis by macrophages engulfing apoptotic cells. Our data reveal a new crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-induced retinoid synthesis in macrophages enhances TG2 expression in the dying thymocytes.

Extracellular matrix molecules and their possible roles in the regeneration of frog nervous system.

Recent biochemical and histochemical analyses explored different components of the extracellular matrix (ECM) in the nervous system, and either permissive or non-permissive roles in neuronal development and regeneration were suggested. The aim of this study was to detect the distribution pattern of a few of these molecules in the nervous system of intact frogs and during nerve regeneration. The hyaluronan (HA) and tenascin C reactions were negative in the peripheral nerves, but appeared in their entry zones. In the CNS, different populations of neurons were surrounded with HA and tenascin C-positive material, forming a perineuronal net (PN). The phosphacan reaction was weakly positive in the PNS, and a moderate intensity was detected in the entry zone and in the PN. Laminin and fibronectin immunoreactivity was strong in the PNS, but laminin could not be detected in the CNS. In animals with cut and regenerating vestibulocochlear nerve, the distribution of the ECM molecules in the CNS and PNS characteristically changed from that of the normal pattern. Our results showed a non-homogenous distribution of ECM components in the frog nervous system that could be associated with their different roles in physiological and pathological processes.

Miller Fisher syndrome--a presenting clinical manifestation of lung cancer in a previously apparently healthy individual.

Sirs: A 54 year-old white male complained of clumsiness of the right upper extremity, diplopia and unsteadiness of gait starting about 6 days before admission. The history was negative except for smoking an average of 2 packs of cigarettes per day in the preceding 40 years. On admission he had right sided complete oculomotor nerve lesion, severe bilateral lower motoneuron facial palsy (Fig. 1A and 1B), absent Achilles reflexes, diminished other deep tendon reflexes and severe gait ataxia. The patient did not have objective sensory loss, major paresis or signs of upper motor neuron lesion. Except for an elevated erythrocyte sedimentation rate (46 mm/hour) and a borderline serum glucose level the routine blood tests were normal. The cerebrospinal fluid (CSF) had an extremely elevated protein content (3.115 g/L) associated with some pleocytosis (256 cells per microliters). The CSF cells were lymphocytes (20 %), macrophages (48 %, a few of them signet-ring cells), monocytes (12 %), and 20 % of the cells were intensely stained, atypical giant cells with large round marginal nuclei. These cells were PAS positive and were also cytokeratin positive with CK7 immunocytochemistry (Fig. 2A). Nerve conduction studies revealed mild axonal sensorimotor neuropathy. On computed tomography contrast enhancement was seen in the right Sylvian fissure (Fig. 2B). MRI detected several small (< 10 mm) contrast enhancing lesions mostly in the cerebral cortex (Fig. 2C, arrows), and in some other CSF-adjacent regions (cerebellar surface, basal ganglia adjacent to the lateral ventricle, periaqueductal gray matter) as well. Chest CT identified a small tumor in the right lung (Fig. 2D). The tumor was removed and appeared to be a carcinoma with adenomatous structure staining with PAS. With immunohistochemical examination the tumor cells were CK7 positive, CK20 negative, exhibited nuclear positivity with TTF-1, and about 5 % of the cells had nuclear positivity with Mib-1. A histological diagnosis of grade 3 bronchial adenocarcinoma was established. Miller Fisher described a syndrome of ophthalmoplegia, ataxia and areflexia as a variant of the Guillain-Barré syndrome [2]. In a series of 50 cases [4] facial palsy was present in about one third of the cases. Although a cerebrospinal fluid (CSF) cell count over 50/μl is rare in Guillain-Barré syndrome, it has been reported in several cases [6]. Axonal, predominantly, sensory neuropathy was found in 5 of 6 patients with Miller Fisher syndrome (MFS) [9]. Therefore, from the clinical signs the diagnosis of MFS could have been considered for our case. In leptomeningeal carcinomatosis the incidence of clinical signs at presentation were 11 % for oculomotor lesion, 11 % for facial nerve involvement and 15–15 % for cerebellar signs and polyradiculopathy [1]. Therefore the probability of the coincidence of the individual signs of MFS is low. There are only 4 published cases where the syndrome was associated with malignant diseases. Leptomeningeal infiltration was described by Guarino et al. [3] in 2 cases. One of them developed the syndrome 6 months after gastrectomy for cancer and the other patient 4 years after thyroidectomy for cancer and 2 years after the diagnosis of acute LETTER TO THE EDITORS

Detection of Carotid Artery Stenosis by In Vivo Duplex Ultrasound: Correlation With Planimetric Measurements of the Corresponding Postmortem Specimens

Background and Purpose— The correct detection and quantification of carotid artery disease are of decisive impact on patient prognosis and adequate treatment. In this study, we evaluated the ability of ultrasonography to detect and to grade carotid artery stenosis through a comparison of the in vivo ultrasound findings with the planimetric analysis of the corresponding postmortem specimens. Methods— Shortly before their death, 59 critically ill neurological patients (mean age, 70 years) were prospectively examined by extracranial and intracranial Doppler sonography and color-coded duplex ultrasound. Carotid stenosis was classified by hemodynamic and morphological ultrasound criteria. Carotid specimens were removed in toto during autopsy. Under standardized conditions, specimens were redistended, sectioned, and histologically processed. Computerized planimetric measurements of the arteries were carried out and compared with the ultrasound findings. Correlation of the ultrasound and postmortem planimetric findings was available in 93 carotid bifurcations. Results— Through both techniques, 46 carotid arteries were found to be normal. Steno-occlusive carotid lesions ranged from 8.5% to 100% lumen reduction. Overall, r =0.96 and adjusted R2=0.90. For the steno-occlusive carotid lesions, r =0.91. Conclusions— Extracranial and intracranial Doppler and color-coded duplex ultrasound permits reliable detection and quantification of carotid artery stenoses and occlusions even under difficult examination conditions in critically ill patients.

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H- thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that pro- tein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal or-ganisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

The distribution pattern of the hyaluronan receptor CD44 during human tooth development

The aim was to investigate the expression pattern of the major cell-surface hyaluronan receptor CD44, as there are no existing data on its presence or absence in human dental structures at different developmental stages. Immunohistochemical localization of CD44 was studied using a monoclonal antibody, H3, that specifically recognizes an epitope in the common backbone of all CD44 isoforms. The dental lamina displayed a strong CD44 signal; the external enamel epithelium was negative. In the coronal region of the tooth germ the presecretory ameloblasts showed an intense reaction whereas the less differentiated inner enamel epithelial cells showed no signal at the cervical loop where they meet the external enamel epithelium. In the stellate reticulum a moderate reaction was detected. The secretory ameloblasts and the stratum intermedium showed a strong cell-surface CD44 signal. A strong signal was also observed on the odontoblasts and their processes. In the pulp, close to the odontoblastic layer, weak labelling was seen in the walls of capillary vessels. The distribution of CD44 in the human tooth germ corresponds to that of hyaluronan in most locations, suggesting that during tooth development this transmembrane protein plays an important part in hyaluronan-mediated events.

Type X collagen in human enamel development: a possible role in mineralization

Although type X collagen is one of the key molecules in endochondral ossification, no data are available on whether it is present in dental structures when mineralization is proceeding. We therefore monitored the appearance of type X collagen in tooth germs of human samples ranging in gestational age from 17-week-old fetuses to 9-week-old newborn. Using immunohistochemistry, ELISA techniques, and Western blotting, we show that type X collagen is present in human tooth germ during enamel maturation. Intense immunohistochemical staining for collagen type X was observed in the enamel and in the apical parts of secretory ameloblast at the bell stage when the dentine and enamel matrix were already under formation. The odontoblasts, the dentine, and the pulp were not stained. In the early (9-week) postnatal stage, the staining for collagen type X in the enamel matrix was diminished, and only a very weak signal could be detected in the secretory ameloblasts. A positive reaction for collagen type X was also observed in ELISA assay of extracts obtained from human embryonic enamel and hypertrophic cartilage samples. The Western blot analysis of the enamel demonstrated that size of the molecule detected by MoAb X53 is characteristic of the type X collagen. This correlates well with our immunohistochemical findings. Based on these data, we propose that type X collagen is one of the candidate molecules present in the enamel matrix that might be involved in mineralization of the enamel.