Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Deep TMS in a resistant major depressive disorder: a brief report.

INTRODUCTION: Repetitive transcranial magnetic stimulation (rTMS) has proven effective. Recently, a greater intracranial penetration coil has been developed. We tested the efficacy of the coil in the treatment of resistant major depression. METHODS: Our sample included seven patients suffering from major depression who were treated using Brainsway's H1-coil connected to a Magstim rapid 2 stimulator. Deep TMS treatment was given to each patient in five sessions per week over a period of 4 weeks. Patients were treated with 120% intensity of the motor threshold and a frequency of 20 HZ with a total of 1,680 pulses per session. RESULTS: Five patients completed 20 sessions: one attained remission (Hamilton Depression Rating Scale (HDRS)=9); three patients reached a reduction of more than 50% in their pre-treatment HDRS; and one patient achieved a partial response (i.e., the HDRS score dropped from 21 to 12). Average HDRS score dropped to 12.6 and average Hamilton Anxiety Rating Scale score dropped to 9.Two patients dropped out: one due to insomnia and the second due to a lack of response. DISCUSSION: Compared to the pooled response and remission rates when treating major depression with rTMS, deep TMS as used in this study is at least similarly effective. Still, a severe limitation of this study is its small sample size, which makes the comparison of the two methods in terms of their effectiveness or side effects impossible. Greater numbers of subjects should be studied to achieve this aim. CONCLUSIONS: An H1 deep TMS coil could be used as an alternative treatment for major depressive disorder.

Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.

To identify and clone genes that encode cell- or tissue-specific secreted and surface proteins, a polyclonal antiserum was raised against a complex mixture of surface or secreted proteins from the target cell, followed by immunodepletion of antibodies that recognize proteins from a nontarget cell or tissue. The depleted antiserum is used to screen bacteriophage cDNA expression libraries. Because of our interest in how adipocytes communicate with other cells, we have used this method to clone cDNAs encoding secreted and plasma membrane proteins that are induced during adipocyte differentiation. We describe several of these, including a novel plasma membrane-associated protein, S3-12.

Studies on leukemia development in the SJL/J strain of mice.

Spontaneous reticulum cell neoplasms developed in 70-80% of intact male and female SJL/J mice at a mean age of 380 days. The neoplasms had a basic histological pattern of a multicellular type-B reticulum cell neoplasm, as designated by Dunn. The tumors were transplanted in isogenic mice. Serial passages of cell suspensions of these tumors were carried on through at least 4 tumor-inducing generations, and the growing transplanted tumors maintained the structure of a reticulum cell neoplasm similar to the original tumor. A histological survey of spontaneous lesions in SJL/J mice revealed different characteristics of reticulum cell neoplasms, some similar to Hodgkin's lesions. Five or ten feedings of 7,12-dimethylbenz-[alpha]anthracene (DMBA) in polyethylene glycol 400 (1 mg DMBA per feeding) to SJL/J mice induced lymphosarcomas in 74-83% of the mice at a mean age of 157-188 days. A single feeding of DMBA induced lymphosarcomas in only 27% of the mice at a mean age of 246 days. These DMBA-induced lymphatic leukemias do not appear to depend on the thymus for development--a 60% incidence of lymphosarcomas was obtained in adult thymectomized mice treated with DMBA, and 27% of the thymectomized DMBA-treated mice developed myeloid leukemia at a mean age of 180 days. Urethan was only slightly leukemogenic in SJL/J mice.

Quantitation, in vitro propagation, and characterization of preleukemic cells induced by radiation leukemia virus.

Intrathymic (i.t.) inoculation of radiation leukemia virus into C57BL/6 mice induces a population of preleukemic (PL) cells that can progress into mature thymic lymphomas upon transfer into syngeneic recipients. A minimum of 10(3) PL thymic cells are required to induce lymphomas in the recipient. Most of the individual lymphomas developed in mice which were inoculated with cells of a single PL thymus, derived from different T-cell precursors. PL thymic cells could be grown in vitro on a feeder layer consisting of splenic stromal cells. Growth medium was supplemented with supernatant harvested from an established radiation leukemia virus-induced lymphoma cell line (SR4). The in vitro-grown PL cells were characterized as Thy-1+, CD4+, CD8- T-cells, most of which expressed radiation leukemia virus antigens. Cultured PL cells were found to be nontumorigenic, based on their inability to form s.c. tumors. However, these cells could develop into thymic lymphomas if inoculated i.t. into syngeneic recipients. A culture of PL cells, maintained for 2 mo, showed clonal T-cell receptor arrangement. Lymphomas which developed in several recipient mice upon injection with these PL cells were found to possess the same T-cell receptor arrangement. These results indicate that PL cells can be adapted for in vitro growth while maintaining their preleukemic character.

Clonal analysis of radiation leukemia virus-induced leukemic and preleukemic murine cells.

Clonality of radiation leukemia virus (RadLV)-induced thymic lymphomas was determined by detection of rearrangements in the genetic locus coding for the beta chain of the T-cell receptor (T beta). Unique T beta rearrangements were detected in four of six lymphomas. Two of the T beta-rearranged thymic lymphomas and two in which such a rearrangement was not detected had a biallelic deletion of C beta 1. With an anti-RadLV monoclonal antibody it was found that 1-2 days after virus inoculation more than one-third of the cells in the thymus were infected by the virus. The frequency of virus-positive cells gradually declined and persisted at 1-2% until the appearance of a clonal lymphoma at which time virtually all the cells in the thymus were virus positive. Transfer of thymocytes from a single, preleukemic mouse 21 days post-virus inoculation into several adoptive recipients resulted in donor-type thymic lymphomas in the majority of the mice. T beta rearrangement analysis revealed that these lymphomas were clonal and derived from different potentially leukemic (preleukemic) cells in the thymus of the donor mouse. Eleven of 15 lymphomas had a biallelic deletion of C beta 1. These results suggest that clonal, RadLV-induced thymomas are selected from an oligoclonal, RadLV-infected preleukemic T-cell population.

Multifocal vascular tumors in fowl induced by a newly isolated retrovirus.

The morphological features of a spontaneous, multifocal vascular neoplasm of chickens are described. Histologically, the tumor was characterized by areas consisting of freely anastomosing vascular channels with prominent papillary appearance and lined by bland-looking endothelial cells, which alternate with areas resembling cavernous hemangioma. Occasionally solid areas composed of plump, pleomorphic cells were also present. Although there was no clear evidence for metastatic spread, some tumors were obviously invasive. Electron microscopy and immunohistochemistry confirmed the endothelial nature of neoplastic cells, demonstrating in particular pinocytic vesicles, well developed junctional complexes, fragmented basal lamina, occasional Weibel-Palade bodies, and patchy factor VIII-related antigen immunoreactivity. The overall appearance of the tumor was that of a cavernous hemangioma with prominent papillary endothelial hyperplasia. Previously we have shown that the tumor was induced by a newly isolated strain of avian hemangioma retrovirus and in this study we demonstrated typical type C retrovirus particles in the tumor by electron microscopy. It is suggested that this retrovirus-induced avian tumor may serve as a useful model for the study of transformed endothelia and other vascular tumorigenesis.

RNAase-sensitive DNA-dependent DNA polymerase from rat cells transformed by avian sarcoma virus.

An RNAase-sensitive DNA polymerase from rat cells transformed by avian sarcoma virus has been characterized. The enzyme requires RNA for its activity, as shown by its sensitivity to RNAase with endogenous as well as exogenous DNA templates. This sensitivity is maintained after its purification by sucrose gradients and ion exchange columns. A molecular weight of about 100 000 has been estimated. This DNA polymerase requires high salt concentration for its activity, is resistant to high concentrations of phosphonoacetic acid (400 micrograms/ml), is partially inhibited by 5 mM N-ethylmaleimide, and is completely inhibited by 0.3 mM parahydroxymercuribenzoate.

Genetic alterations of integrated avian sarcoma virus DNA sequences in transformed rat cells.

Clones and subclones of Schmidt-Ruppin-RSV-D-(SRD-) infected rat cells that were isolated in soft agar have shown differences in their morphology. Some of the subclones were round or spindle-shaped cells with lower anchorage dependence and high growth rate, while others were more fibroblast-like and failed to overgrow one another. In correlation to their morphology, subclones with high degree of phenotypic transformation contained amplified amounts of full length proviral DNA or proviral fragments. The amplification affected the proviral DNA elements together with host cellular DNA sequences. We have also shown a relationship between the copy number of the proviruses in the cells, the level of expression of viral RNA, and between those two parameters and the various degrees of phenotypic transformation.

The methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells.

The endogenous viruses in the avian cells are not completely methylated, nor are the Schmidt-Ruppin RSV-D (SRD) proviruses in the infected cells completely unmethylated. Avian sarcoma proviruses integrated in rat transformed cloned cells are heavily methylated. In these cells, a region in the 3' end of the env gene is unmethylated in all the src-containing proviruses but not in the transformed defective (td) proviruses. A possible role for the hypomethylation of the 3' end of the env region is proposed.

Exercise systolic blood pressure: a powerful determinant of increased left ventricular mass in patients with hypertension.

This study examines the relation between left ventricular mass determined by two-dimensional echocardiography and exercise blood pressure in patients with hypertension. Sixty-seven patients with hypertension and 19 normal subjects underwent treadmill exercise testing and two-dimensional echocardiography. The left ventricular mass index in the normal subjects was 80 +/- 10 g/m2 (mean +/- SD). Patients with hypertension were classified into two groups according to left ventricular mass: Group I (n = 42) had normal mass and Group II (n = 25) had increased mass (greater than 2 SD above the mean value in 19 normal subjects). There was a poor correlation between left ventricular mass and blood pressure at rest. However, a better correlation was found between left ventricular mass and exercise systolic blood pressure (r = 0.58, p less than 0.001) or the change in systolic blood pressure from rest to exercise (r = 0.48, p less than 0.001). Twenty-two (76%) of 29 patients with an exercise systolic blood pressure of 190 mm Hg or greater had an increased left ventricular mass index, whereas only 3 (8%) of 38 patients with an exercise systolic blood pressure of less than 190 mm Hg had an increased left ventricular mass index (p less than 0.0001). Thus, in patients with hypertension, left ventricular mass index is poorly related to blood pressure at rest, but is related to exercise systolic blood pressure. Patients with an exercise systolic blood pressure of 190 mm Hg or greater usually have an increased left ventricular mass. These findings may have therapeutic implications.

Right atrial compression in postoperative cardiac patients: detection by transesophageal echocardiography.

Four patients developed hypotension after heart surgery. Hemodynamic measurements revealed elevated right atrial pressure with normal pulmonary capillary wedge pressure. Conventional transthoracic two-dimensional echocardiography was technically suboptimal for detection of pericardial effusion. In each patient transesophageal echocardiography demonstrated significant compression of the right atrium by a localized mass. At reoperation atrial compression by an organized hematoma was found and in each instance successfully drained. Thus, transesophageal echocardiography is superior to transthoracic echocardiography in evaluating critically ill postoperative hypotensive patients and can differentiate isolated right atrial tamponade from other causes of hemodynamic deterioration such as prosthetic valve dysfunction or left ventricular systolic dysfunction, or both.

Flow characteristics in the dilated left ventricle with thrombus: qualitative and quantitative Doppler analysis.

In an attempt to determine whether mural thrombus in a dilated left ventricle is associated with specific flow patterns, a study was undertaken to assess qualitatively and quantitatively the flow characteristics by conventional pulsed and two-dimensional Doppler color flow examination. Forty patients with cardiomyopathy formed the study group (20 with an apical thrombus and 20 without). The groups did not differ with respect to age, gender, origin of ventricular dysfunction, ventricular size and ejection fraction. Guided by the Doppler color flow pattern, a quantitative analysis of flow velocity profile in the ventricular inflow and outflow "compartments" was performed by serial pulsed wave Doppler sampling. Inflow velocity at the ventricular apex was significantly lower in the thrombus group than in the no thrombus group (11.7 +/- 15.3 versus 28.3 +/- 10.5 cm/s, p less than 0.0001). Flow velocities were generally lower in the thrombus group at the other levels in the inflow compartment (that is, mitral anulus, leaflet tips and papillary muscle level). The systolic flow velocity at the apex was similarly significantly lower in the thrombus group than in the no thrombus group (7.1 +/- 8.1 versus 15.3 +/- 7.0 cm/s, p less than 0.001). Additionally, a higher prevalence of mitral regurgitation was noted in the no thrombus group (14 patients) than in the thrombus group (3 patients). Thus, specific abnormal flow profiles are associated with a left ventricular thrombus. Whether the abnormal flow is a primary event in the genesis of left ventricular thrombus or occurs secondary to development of the thrombus cannot be determined from this study.

Two-dimensional echocardiographic estimation of right ventricular ejection fraction in patients with coronary artery disease.

Two-dimensional echocardiographic determination of right ventricular ejection fraction was compared with right ventricular ejection fraction obtained by first pass radionuclide angiography in 39 patients with coronary artery disease. Apical four chamber and two chamber right ventricular views were obtained in 34 (87%) of the 39 patients, while a subcostal four chamber view was obtained in 31 patients (80%). Right ventricular ejection fraction by two-dimensional echocardiography was calculated by the biplane area-length and Simpson's rule methods using two paired orthogonal views and utilizing a computerized light-pen method for tracing the right ventricular endocardium. A good correlation (r = 0.74 to 0.78) was found between radionuclide angiographic and two-dimensional echocardiographic right ventricular ejection fraction for each method used. Patients with acute inferior myocardial infarction had the lowest right ventricular ejection fraction by radionuclide angiography and two-dimensional echocardiography (p less than 0.05 compared with patients with right coronary artery obstruction and no infarction). There were no differences in right ventricular ejection fraction between patients with acute and old inferior myocardial infarction by both techniques. No correlation was found between left and right ventricular ejection fraction by radionuclide angiography (r = 0.16). It is concluded that 1) right ventricular ejection fraction by two-dimensional echocardiography correlates well with radionuclide angiographic measurements and can reliably evaluate right ventricular function in coronary artery disease, 2) patients with inferior myocardial infarction have reduced right ventricular ejection fraction, and 3) changes in left ventricular ejection fraction do not directly influence right ventricular function.

Noninvasive evaluation of normal and abnormal prosthetic valve function.

Noninvasive techniques are helpful in evaluating the function of mechanical prostheses and tissue valves. Combined phonocardiography and M-mode echocardiography together with cinefluoroscopy are the most useful noninvasive techniques in differentiating normal from abnormal metallic prosthetic valve function. The intensity of the opening and closing clicks and associated murmurs will depend on the type of prosthetic valve, the heart rate and rhythm and the underlying hemodynamic status. Arrhythmias or conduction disturbances, or both, may produce motion patterns that mimic some of the echocardiographic signs of malfunctioning prosthetic valves. Differentiation of thrombus formation or tissue ingrowth from paravalvular regurgitation or dehiscence is possible by noninvasive techniques. Disc variance, a potentially serious and lethal problem with the older Beall valves, can be readily detected by cinefluoroscopy and echophonocardiography. With regard to bioprosthetic valves, two-dimensional echocardiography is superior to M-mode echocardiography in detecting primary valve failure. In addition, detection of vegetations, valve alignment and ring and individual leaflet motion can be best accomplished by two-dimensional echocardiography. Of greater importance is the patient serving as his or her own control in the follow-up assessment of prosthetic valve function by noninvasive techniques.

Development and regression of left ventricular hypertrophy.

Left ventricular hypertrophy is an important adaptive response to chronic pressure or volume overload of the left ventricle. The different types and the pathophysiologic mechanisms of the development of left ventricular hypertrophy in various disease states are reviewed. Detection of left ventricular hypertrophy may be accomplished by electrocardiography and cardiac angiography. Echocardiography, however, is the most accurate noninvasive method to detect the presence and estimate the severity of increased left ventricular mass. The clinical significance of left ventricular hypertrophy and its prognostic implications in several cardiac diseases associated with hypertrophy are discussed. The critical transition stage from adaptive, compensatory and reversible left ventricular hypertrophy to "pathologic" hypertrophy with impaired left ventricular contractility and irreversible myocardial damage is yet unknown. Recent data are presented that provide evidence of regression of left ventricular hypertrophy after medical treatment of patients with hypertension and after aortic valve replacement in patients with aortic valve disease. The clinical importance of regression of hypertrophy and its effects on long-term prognosis remain to be determined.

Cardiac function in patients with cystic fibrosis: evaluation by two-dimensional and Doppler echocardiography.

Two-dimensional and Doppler echocardiography were performed in 17 consecutive hospitalized patients with cystic fibrosis aged 6 to 38 years (mean 21 +/- 9) and in 10 normal subjects aged 24 +/- 7 years. Left ventricular and right ventricular ejection fraction were measured by a computerized light pen system and Simpson's rule from two-dimensional echocardiographic apical four and two chamber views. Right ventricular wall thickness, inferior vena cava size and the presence of tricuspid regurgitation by Doppler recording were also assessed. National Institutes of Health (NIH) score of clinical severity ranged from 22 to 72 (mean 51 +/- 15) (100 = excellent, 0 = poor). Four patients, all with an NIH score of 40 or less, died of respiratory failure within 1 year of the echocardiographic study. There was no significant difference between patients with cystic fibrosis and normal subjects with regard to right ventricular ejection fraction (59 +/- 11 versus 61 +/- 10%), left ventricular ejection fraction (67 +/- 8 versus 70 +/- 8%) and right ventricular systolic (5 +/- 1 versus 5 +/- 0.5 mm) and diastolic (2.4 +/- 0.5 versus 2.5 +/- 0.5 mm) wall thicknesses. A dilated inferior vena cava and mild tricuspid regurgitation by Doppler recording were detected in only one patient. A poor correlation was found between right ventricular ejection fraction and NIH clinical score (r = 0.26), chest X-ray score (r = 0.29) and pulmonary function tests. It is concluded that right and left ventricular systolic function is preserved in patients with moderately severe cystic fibrosis; clinical status in these patients is probably determined by the pulmonary rather than cardiac involvement.

Two-dimensional echocardiographic determination of left atrial emptying volume: a noninvasive index in quantifying the degree of nonrheumatic mitral regurgitation.

Several noninvasive techniques, including radionuclide angiography and Doppler echocardiography, have attempted to measure the regurgitant volume in patients with mitral regurgitation; however, none of these techniques are entirely satisfactory. Utilizing a computerized light pen method for tracing the left atrial endocardial border during systole and diastole in two orthogonal planes (apical four and two chamber views), biplane volume determinations were calculated in 12 normal subjects and 30 patients with nonrheumatic mitral regurgitation. Left atrial emptying volume determinations were performed by subtracting the left atrial end-diastolic volume from the left atrial end-systolic volume. The degree of mitral regurgitation was visually assessed as normal (0, trivial, Group I, 12 patients), mild (1+, Group II, 4 patients), moderate (2+, Group III, 8 patients), moderately severe (3+, Group IV, 12 patients) and severe (4+, Group V, 6 patients) by contrast left ventricular angiography and also quantitatively by regurgitant fraction at cardiac catheterization. All 18 patients with moderately severe (Group IV) and severe (Group V) mitral regurgitation had a left atrial emptying volume greater than 40 ml compared with none of the normal subjects and patients with mild (Group II) or moderate (Group III) mitral regurgitation. There was good correlation between left atrial emptying volume and mitral regurgitant fraction (r = 0.85, p less than 0.01). Thus, in patients with nonrheumatic mitral regurgitation, left atrial emptying volume is useful in separating mild from severe mitral regurgitation.

Tricuspid regurgitation: noninvasive techniques for determining causes and severity.

Tricuspid regurgitation is often not apparent on physical examination and several methods are now available to aid in this difficult assessment. Cardiac catheterization using right ventriculography, previously considered the diagnostic standard, has several limitations. Currently available noninvasive tools such as M-mode and two-dimensional echocardiography (with or without contrast), Doppler techniques and even radionuclide cardiologic imaging have added significantly to the precise assessment of the presence and severity of tricuspid regurgitation. This review examines the comparative use and limitations of these various techniques.