The efficacy of commercial tooth storage media for maintaining the viability of human periodontal ligament fibroblasts.

AIM: To evaluate Save-A-Tooth (SAT), EMT Toothsaver (EMT) and Hank's Balanced Salt Solution (HBSS) for their influence on the viability and proliferative capacity of human periodontal ligament fibroblasts (HPDLFs). METHODOLOGY: Primary HPDLFs were seeded into 96-well cell culture plates and exposed to SAT, EMT, HBSS and water (negative control) for 0.5, 1, 3, 6, 12 and 24 h at room temperature (22 °C). After each exposure time, cell viability was measured through quantifying adenosine triphosphate (ATP) using a luminescent dye. The proliferative capacity was also quantified using the PrestoBlue assay after 12 or 24 h storage in each medium. The data were analysed statistically by two-way anova and post hoc Least Significant Difference (LSD) test (P < 0.05). The morphology of the cells after 12 h storage was also investigated through live/dead viability/cytotoxicity kit together with fluorescence microscopy. RESULTS: There was no significant difference in cell viability amongst HBSS, SAT and EMT groups up to 6 h. SAT was effective in maintaining cell viability only up to 12 h and then became detrimental to HPDLF; after 24 h, the effectiveness of SAT in maintaining cell viability was similar to that of water (P > 0.05). Amongst all the media, only EMT could maintain the proliferative capacity of HPDLFs significantly higher than the negative control, that is water (P < 0.05) after 24 h storage. CONCLUSION: EMT maintained the proliferative capacity of HPDLFs after 24 h storage.

Rare cardiomyopathies: diagnostic features.

Cardiomyopathies (CM) are an important and heterogeneous group of diseases affecting the myocardium. They can induce mechanical and/or electrical disorders and are due to a variety of causes, they frequently are genetic. However, since their high number and their clinical complexity, the identification is still a challenge. Echocardiography is a very useful tool in the assessment of CM. In this review we aim to define the typical clinical features and to discuss the main diagnostic tool, above all echocardiography that can help physicians in the correct assessment of CM.

Genetic and epigenetic defects at the 6q24 imprinted locus in a cohort of 13 patients with transient neonatal diabetes: new hypothesis raised by the finding of a unique case with hemizygotic deletion in the critical region.

BACKGROUND: Transient neonatal diabetes (TND) is a rare form of diabetes usually present in the first few days after birth that resolves within 1 year but that has a tendency to recur later in life. It can be associated with chromosome 6 paternal uniparental disomy (UPD), paternal duplications or loss of maternal methylation at the 6q24 imprinted locus. OBJECTIVE: To report on a cohort of 13 sporadic TND cases, including five with birth defects (congenital abnormalities of heart, brain and bone) and eight without. RESULTS: The hallmarks of diabetes were similar in patients with or without 6q24 defects. The chromosome 6 abnormalities in our patients (n = 13) included 2 of 13 (approximately 15.4%) cases of paternal UPD6, 2 of 11 (approximately 18%) cases of complete and 3 of 11 (approximately 27%) cases of partial loss of the maternal methylation signature upstream of ZAC1-HYMAI imprinted genes in non-UPD cases, and 1 of 13 (approximately 7.7%) cases of hemizygotic deletion. CONCLUSION: The deletion was found in a patient with severe congenital abnormalities. This genetic lesion was not reported previously. The hypothesis of an effect on regulatory elements critical for imprinting and tissue-specific gene expression in early development by the deletion is raised. The data presented here may contribute to the diagnosis and the understanding of imprinting in the region.

The role of echocardiography in the evaluation of cardiac damage in hypertensive obese patient.

Obesity rates are rising worldwide. Often obesity is associated with hypertension leading to an increased cardiovascular risk. Both obesity and hypertension induce several modifications in cardiac structure and function, particularly atrial and ventricular remodeling is a common finding shared by these two conditions. It has been demonstrated obesity leads to: left ventricular (LV) mass increase, LV systolic and diastolic dysfunction, left atrium (LA) size increase, LA function alterations and pericardial fat accumulation. Nowadays, the development of cardiac imaging techniques allows to early identifying any preclinical damage related to hypertension and obesity. This could be very important in order to improve patient management and medical therapy.

Variable features of transient neonatal diabetes mellitus with paternal isodisomy of chromosome 6.

We describe two patients who suffered transient neonatal diabetes mellitus (TDNM), due to paternal isodisomy of chromosome 6. One patient, now 5 years old, had severe intra-uterine growth retardation, but recovered normal growth parameters. The other patient, currently 12 years old, had a normal birth weight but showed impaired post-natal growth; in addition to TNDM the patient presented with cardiac and thyroid abnormalities. These cases may suggest that the clinical phenotype of TNDM is more variable than previously believed. The contribution of genetic and epigenetic factors needs to be determined to elucidate the phenotype-genotype relationships of this disease.

Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.

Dynamin (Dyn) is a member of a novel group of GTPases which was initially identified as a microtubule-binding protein with a role in vectorial movement. Three distinct Dyn-encoding genes (DYN I, II and III), with a neuronal-, ubiquitous or testis-specific expression, respectively, have been identified in rat. In man, only DYN I has so far been characterized. We have previously isolated a genomic DNA fragment implicated in the correction of mitomycin C hypersensitivity of cells from a Fanconi anemia patient belonging to genetic complementation group D (FA(D)). Using this probe, we have cloned a human complementary DNA designated hDYN II encoding a ubiquitous Dyn isoform. The predicted protein consists of 866 amino acids (97.5 kDa). Dyn proteins exhibit a high degree of evolutionary conservation: hDyn II is 98% identical to rat Dyn II and 73% identical to hDyn I. A unique 3.6-kb transcript is found in all human tissues examined and it is more abundant in skeletal muscle and heart. This transcript is also expressed in tissue-culture cells. The hDYN II message is present and not mutated in the FA(D) patient studied. In addition to the GTP-binding domain and motifs associated with regulatory function, the hDyn II protein contains a noticeable number of concensus motifs for p34Cdc2 kinase phosphorylation which may indicate a potential role at the G2/mitosis transition. The sequence reported here should allow a more complete analysis of Dyn function(s) in man.

Severe or marginal iron deficiency affects spontaneous physical activity in rats.

The influence of dietary iron on spontaneous activity was investigated in growing rats. Male rats weighing 97 +/- 10 g fed diets containing 4.6 (deficient), 19.9 (marginal), and 108.4 (adequate) mg Fe/kg diet for 8 wks had hemoglobin values of 52 +/- 6, 152 +/- 4, and 159 +/- 6 g/L, and liver nonheme iron concentrations of 0.79 +/- 0.24, 1.36 +/- 0.39, and 4.58 +/- 0.62 mumol/g, respectively. Body weights were affected only at the lowest iron intake. Diurnal activity was not reversed by iron status; all animals were more active during the dark cycle than the light cycle. Iron deficiency resulted in less time and frequency of horizontal, vertical, and stereotypic movements; less distance moved; and less frequent rotations. Movement speed of iron-deficient rats was faster during early iron deficiency. In the light, rats with marginal iron nutriture were generally more active than were iron-deficient or iron-adequate animals. In the dark, activity generally decreased with each reduction in dietary iron.

Effect of low dose rate ionizing radiation on the division potential of cells in vitro. VIII. Cytogenetic analysis of human fibroblasts.

Human embryonic and adult cells were irradiated with fractionated doses of low dose rate ionizing radiation starting early during their lifespan. Adult cells were found to be more sensitive than fetal cells to ionizing radiation in terms of the number of cells produced during the lifespan of the control and the irradiated cultures. Phase-III adult control cells had fewer chromosomal aberrations than phase-III embryonic control cells. After irradiation there was an increase in chromosomal aberrations in adult cells but no increase in embryonic cells beyond those found in the control cultures. It is suggested that cells that have a higher potential for chromosomal rearrangements survive better after low dose rate ionizing radiation.

An abnormal expression of a tumor-activated multigenic set in cells from cancer prone patients with inherited Fanconi's anemia (FA) and retinoblastoma (Rb).

Normal skin fibroblasts from patients genetically predisposed for cancer express certain phenotypic properties usually associated with transformed cells, suggesting that these cells are engaged in a cancer progression process. Previously, we have shown that a subgenomic fraction, which we called Ta DNA ('tumor-activated DNA') was preferentially expressed in all human tumor cells (Hanania et al., Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 6504-6508; Hanania et al, EMBO J., 2 (1983) 1621-1624). Here, we show that Ta DNA transcripts are present in Fanconi's anemia (FA) and retinoblastoma (Rb) fibroblasts, at a level hardly lower than in tumor cells. In contrast, a few Ta DNA transcripts can be detected in normal cells.

Biochemical and behavioral anxiolytic-like effects of R(+)HA-966 at the level of the ventral tegmental area in rats.

RATIONALE: R(+) HA-966, a weak partial agonist at the glycine/NMDA receptor complex, has been shown to have anxiolytic-like actions on restraint stress-induced mesoprefrontal dopamine metabolism. OBJECTIVE: This study investigates the putative anxiolytic, R(+) HA-966, applied locally at the level of the mesocorticolimbic dopamine cell bodies in the ventral tegmental area (VTA), on the acquisition and expression of conditioned fear. METHODS: Ten to 14 days after cannula implantation, rats were subjected to the acquisition session (10x5 s tone paired with 0.5 s, 0.8 mA footshock) followed about 24 h later by the expression session (ten tones only) of a conditioned fear protocol. Rats were treated with R(+) HA-966 (15 microg/VTA) or saline before either the acquisition or expression sessions. Other rats were injected with saline or R(+) HA-966 (10 microg/side), intra-medial prefrontal cortex, on the expression day. RESULTS: R(+)HA-966, intra-VTA, prevented stress-induced changes in mesoprefrontal, but not mesoaccumbal, dopamine metabolism and was associated with a reduction in fearful responses to physical (footshock) and psychological (conditioned fear) stressors. Additionally, rats treated with R(+)HA-966 intra-VTA before the acquisition session were less fearful at the beginning of the expression session. Local injection of R(+)HA-966 into medial prefrontal cortex did not have anxiolytic-like behavioral or biochemical actions but diminished the expression of exploratory behavior in non-stress, control rats. CONCLUSIONS: These studies indicate that the stress-induced activation of the mesoprefrontal dopamine neurons is necessary for the normal expression of fearful behaviors.

Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation.

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.

Identification and chromosomal localization of a DNA fragment implicated in the partial correction of the Fanconi anemia group D cellular defect.

Fanconi anemia (FA) cells, complementation group D, which had been transfected with mouse genomic DNA were partially corrected for their mitomycin C (MMC) hypersensitivity. A genomic DNA fragment which complements the resistance of FA(D) cells to MMC close to normal level has been cloned; it has no correcting activity in FA group A cells. It contains two highly conserved regions between the mouse and human genome, which flank mouse repeated DNA. This DNA fragment detects a 3.6-4-kb mRNA transcript in human cells. Moreover this fragment maps to chromosome 11q23, a region of particular interest since several genes involved in the control of major cellular functions are located in this area. This DNA fragment may belong to a gene directly or indirectly involved in FA(D) function.

[Cardiologic diagnosis of pulmonary embolism: echocardiography]. / Diagnostica cardiologica dell'embolismo polmonare: l'ecocardiografia.

Pulmonary embolism (PE) represents the third more frequent cardiovascular disease following the acute coronary artery disease and stroke. The most important predisposing clinical condition for PE is represented by the deep-vein thrombosis. The clinical diagnosis of PE has a very low accuracy; so the clinical suspect has to be necessarily directed towards the performance of diagnostic procedures. Among the most used procedures, the echocardiography has a diagnostic role but also a prognostic one. Moreover, it offers precious informations useful to perform the most suitable treatment. The echocardiography features which suggest the presence of pulmonary embolism are: right ventricle and atrium dilatation, right ventricular hypokinesia, systolic flattening of the interventricular septum, tricuspid regurgitation, pulmonary artery dilatation, disappearance or reduction of the inspiratory collapse of the inferior vena cava and presence of eventual embolic sources. According to the involvement degree of right ventricular function, it is generally possible to identify a different survival. The subgroup of patients with moderate or severe right ventricular dysfunction shows a high in hospital and within 1 year death rate. For this reason the right ventricular dysfunction degree together with the hemodynamic stability, are the most important parameters in the therapeutic choice. If there is no right ventricular dysfunction a treatment with heparin is indicated. In presence of right ventricular dysfunction and hemodynamic instability, the thrombolytic treatment is necessary. If the patient is hemodynamically stable, a transesophageal echocardiography is recommended; in case of central thrombosis the thrombolytic therapy or surgery are needed, while if no embolic material is shown the heparin treatment is advisable.

[Thrombo-embolic risk evaluation in patients with atrial fibrillation. Role of echocardiography]. / Stratificazione del rischio tromboembolico nel paziente con fibrillazione atriale. Ruolo dell'ecocardiografia.

Atrial fibrillation (AF) is the most common observed cardiac arrhythmia and is the most frequent condition associated with thromboembolic events in patients with or without mitral valve disease. The source of cardiac emboli is the left atrium, the left atrial appendage or, less frequently, the left ventricle. Emboli may also originate from aortic atherosclerotic plaques. It is important to identify patients at risk in order to perform the appropriate therapy. Risk stratification is multiparametric, being based on clinical, laboratory, and echocardiographic data. Several trials have pointed out the role of echocardiography in the evaluation of anatomic and functional parameters associated with thromboembolic risk. Transthoracic echocardiography (TTE) does not provide sufficient information regarding posterior cardiac structures, being its sensitivity in detecting thrombi relatively low (33-72%). Transesophageal echocardiography (TEE) in contrast, has an almost 100% sensitivity; this technique is, therefore, mandatory in patients with AF for an adequate prevention of thromboembolism. The echocardiographic information joined with clinical features allow to stratify, in a proper way, the risk of every single patient.

[Detection of left ventricular endocardial border with tissue harmonic imaging]. / Vantaggi della seconda armonica tissutale nella identificazione del bordo endocardico ventricolare sinistro.

BACKGROUND: Ultrasonic wave propagation in human tissues is not linear. As a consequence, harmonic waves, whose frequency is a multiple (harmonic) of the emitted frequency, are generated. Tissue Harmonic Imaging (THI) processes only the second harmonic frequency in order to improve the signal-to-noise ratio of the received signal. The study was aimed at investigating the impact of THI on the detection of the Left Ventricular (LV) endocardial border. Attention was paid to determine which LV walls were analysed more clearly with THI rather than with conventional Fundamental Frequency Imaging (FFI). METHODS: We compared the FFI and the THI visualization of the 16 LV segments and of the 6 LV walls in 30 consecutive patients by scoring the images as bad, sufficient or good. The equipment used was a GE Vingmed System Five (Orten, Norway). RESULTS: Images were good in 85% of segments explored with THI, whereas FFI provided good images in 47% of segments (p<0.001). From the apical window, visualization of the apex, lateral wall and anterior wall significantly improved with THI; using the parasternal approach, imaging of posterior wall was definitely better with THI than with FFI. CONCLUSIONS: THI enhances both endocardial visualization and global image quality. Tech-nical development, however, increases the diagnostic possibilities of echocardiography but does not substitute the operator's experience.

[The second tissue harmonic signal: from physics principles to clinical application]. / La seconda armonica tissutale: dai principi fisici all'applicazione clinica.

The second harmonic signals received from organs are due to the non linear properties of tissue which cause distortion of the transmitted signal and are not primarily caused by the transmission of a harmonic frequency. The velocity of ultrasound propagation depends on the density of the insonified material. During the compression phase, the tissue becomes denser, and the ultrasound waves travel faster through the tissue than during the rarefaction phase; the compression phase tends to overtake the rarefaction phase. The ultrasound waveform thus, undergoes a distortion that becomes greater as the distance from the transducer increases. Due to these effects, the tissue tends to generate harmonics and hence shifts energy from the fundamental to the harmonic bands. There are several reasons why harmonic tissue imaging increases the signal-to-noise ratio and facilitates interpretation. In technically difficult patients, there is often a diffuse haze due to distortion of the transmitted beam by shallow surface layers or to reverberations between the skin and ribs. These distortions and reverberations consist almost entirely of ultrasound energy at the fundamental frequency. When the returned signal is filtered at the harmonic so as to reject the fundamental frequency, the clutter and haze are removed and the image becomes more clear and defined. A further reason for the decrease in artifacts and clutter is the side-lobe level reduction in the second harmonic beam. Thus, harmonic beams are narrower and have lower side-lobe levels than fundamental ones. There are several clinical applications of harmonic tissue imaging. These include the correct definition of endocardial borders resulting in an improved assessment of left ventricular function at rest as well as during stress testing, the delineation of the left atrial appendage, the detection of atrial right to left shunting, and left atrial spontaneous echo contrast. Moreover, improved endocardial visualization leads to better endocardial tracking with acoustic quantification and to more segments being interpretable with the anatomic M-mode.

PD-L1 Expression in Clear Cell Renal Cell Carcinoma: An Analysis of Nephrectomy and Sites of Metastases.

BACKGROUND: Expression of programmed death ligand (PD-L1/B7-H1/CD274) represents a mechanism of immune escape for renal cell carcinoma (RCC) cells. Drugs blocking PD-L1 or its receptor are in clinical development and early data suggests that tumor PD-L1 expression may predict response. PATIENTS AND METHODS: A tissue microarray (TMA) consisting of four biopsy cores from 34 matched pairs of nephrectomy and metastatic sites of clear cell RCC was used to assess PD-L1 expression by quantitative immunofluorescence. Assessment of intra- and inter-tumor heterogeneity and primary and metastatic tumor expression was performed using a method of Automated Quantitative Analysis (AQUA). RESULTS: The median AQUA scores were higher in metastatic than primary specimens (P < 0.0001). The correlation between PD-L1 expression in matched primary and metastatic specimens was weak (R= 0.24). Within a given tumor, variable PD-L1 staining heterogeneity was seen, however the degree of heterogeneity was similar in primary and metastatic sites (P = 0.482). CONCLUSIONS: The weak correlation between PD-L1 expression in primary and metastatic sites for a given patient suggests that expression in nephrectomy specimens cannot be used to select metastatic RCC patients for PD-L1 and PD-1 inhibitors. The intra-tumor heterogeneity seen in both primary and metastatic specimens indicates that a single core biopsy might not be sufficient to determine PD-L1 expression.

Calcified disease of the mitral annulus: a spectrum of an evolving disease.

Mitral annulus calcification may appear under different forms depending from its evolution stage: mitral annulus calcification; homogeneous calcified mass of the mitral valve; liquefaction necrosis of the mass; reduction or stability of the mass dimension. We report a large calcified mass located in between the posterior mitral valve leaflet and adjacent left ventricular myocardium suggesting the homogeneous calcified phase of the disease.