The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food.

AIM: To develop antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food products. METHODS AND RESULTS: Aptamer, a single-stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer-A8, specific for internalin A, an invasive protein of L. monocytogenes, was used in the fibre-optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti-Listeria antibody, P66, was immobilized on streptavidin-coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647-conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10(3) CFU ml(-1). This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10(2) CFU 25 g(-1) ) ready-to-eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. CONCLUSION: Based on the data presented in this study, the antibody-aptamer functionalized fibre-optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates feasibility and novel application of aptamer on fibre-optic biosensor platform for the sensitive detection of L. monocytogenes from food products.

Tubulointerstitial nephritis with IgM-positive plasmacytoid large lymphocyte infiltration in a patient with primary biliary cirrhosis and Sjögren's syndrome.

We report a 38-year-old woman diagnosed with tubulointerstitial nephritis (TIN) on renal biopsy, followed by being diagnosed with primary biliary cirrhosis (PBC) and Sjögren's syndrome (SS). Immunohistochemically, the cellular infiltrates in TIN were mainly composed of small lymphocytes and IgM-positive plasmacytoid large lymphocytes. IgM-positive plasmacytoid large lymphocytes were not identical with, but colocalized with CD3- or CD20-positive lymphocytes. TIN in patients with PBC is very rare and little is known about immunohistochemical characteristics of infiltrating cells in this setting. To our knowledge, this is the first report demonstrating predominant infiltrating of IgM-positive plasmacytoid large lymphocytes in TIN due to PBC and SS.

Seizure discharges evoked in vitro in thin section from guinea pig hippocampus.

A thin section prepared from guinea pig hippocampus produced, in chloride-free medium, a train of seizure discharges in response to a single shock applied to the section. Generation of these discharges was ascribed to the lack of inhibitory processes in an absence of chloride ion.

Acute on chronic subdural hematoma as a rare complication in a microscopic polyangiitis patient receiving antithrombotic treatment.

We report a 56-year-old man with microscopic polyangiitis (MPA) who developed acute exacerbation of a chronic subdural hematoma (SDH). Laboratory data demonstrated elevation of myeloperoxidase antineutrophil cytoplasmic antibody (MPOANCA) and rapidly progressing renal dysfunction. Renal biopsy showed crescentic glomerulonephritis (GN) with membranous nephropathy (MN). He was treated with corticosteroids, antithrombotic agents, and an immunosuppressant. One month after initiation of treatment, he had a mild headache. One month later, he developed acute SDH. Although he recovered completely after the operation, he finally died of bacterial infection. On autopsy, a scar of vasculitis was confirmed in the leptomeninges as well as in the kidney and lung. Although SDH is a rare complication in MPA, nephrologists must pay more attention to the initial symptoms before a hematoma attack such as headache, especially in patients using antithrombotic agents.

[Listeria monocytogenes: report of a rise in pregnant women and literature review]. / Listeria monocytogenes: Informe de un aumento de casos en mujeres embarazadas y revisión de la literatura.

Listeria monocytogenes, rare pathogen in the general population, causes serious infections in patients at the extreme ages of life, pregnant woman, and those with immunosuppression. The clinical manifestations are essential to suspect the disease in patients at risk, allowing an early prescription of antimicrobial therapy, before the results of the cultures are available. Clinical course and prognosis depends on how early treatment is started and, in pregnant women, the gestational age. In Clínica Alemana, at Santiago, we detected a 15 fold rate rise of neonatal listeriosis between year 2007 and 2008. Ten cases were diagnosed between January and July 2008 and the seven cases occurring in pregnant women are reported here. All these patients were in their first pregnancy, which could be associated with similar lifestyle and food habits. Considering this new epidemiological scenario, it is important to educate the population, and to conduct an epidemiological study in order to determine the national situation of Listeria monocytogenes infection.

Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.

We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype interferon sensitivity determining region appears to be interferon-resistant strains. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment.

The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation.

AML-1B is a hematopoietic transcription factor that is functionally inactivated by multiple chromosomal translocations in human acute myeloblastic and B-cell lymphocytic leukemias. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain of AML-1B, with ETO, a nuclear protein of unknown function. We previously showed that AML-1-ETO is a dominant inhibitor of AML-1B-dependent transcriptional activation. Here we demonstrate that AML-1-ETO also inhibits C/EBP-alpha-dependent activation of the myeloid cell-specific, rat defensin NP-3 promoter. AML-1B bound the core enhancer motifs present in the NP-3 promoter and activated transcription approximately sixfold. Similarly, C/EBP-alpha bound NP-3 promoter sequences and activated transcription approximately sixfold. Coexpression of C/EBP-alpha with AML-1B or its family members, AML-2 and murine AML-3, synergistically activated the NP-3 promoter up to 60-fold. The t(8;21) product, AML-1-ETO, repressed AML-1B-dependent activation of NP-3 and completely inhibited C/EBP-alpha-dependent activity as well as the synergistic activation. In contrast, the inv(16) product, which indirectly targets AML family members by fusing their heterodimeric DNA binding partner, CBF-beta, to the myosin heavy chain, inhibited AML-1B but not C/EBP-alpha activation or the synergistic activation. AML-1-ETO and C/EBP-alpha were coimmunoprecipitated and thus physically interact in vivo. Deletion mutants demonstrated that the C terminus of ETO was required for AML-1-ETO-mediated repression of the synergistic activation but not for association with C/EBP-alpha. Finally, overexpression of AML-1-ETO in myeloid progenitor cells prevented granulocyte colony-stimulating factor-induced differentiation. Thus, AML-1-ETO may contribute to leukemogenesis by specifically inhibiting C/EBP-alpha- and AML-1B-dependent activation of myeloid promoters and blocking differentiation.

Method of identifying inhibitors of oncogenic transformation: selective inhibition of cell growth in serum-free medium.

We developed a new method for evaluating inhibitors of oncogenic signal transduction pathways based on different growth abilities between normal and transformed cells in a defined serum-free medium. The growth rates of src, abl or ras oncogene-transformed cells, activated raf proto-oncogene transformed cells, and normal NIH-3T3 cells were 60-90%, 20-30% and 10% in a serum-free medium, respectively, compared to the growth rates in a serum-containing medium. An addition of a growth factor (PDGF, FGF or TGF-beta) stimulated the growth of normal NIH3T3 cells by 40-80% in a serum-free medium. Herbimycin A, a specific cytoplasmic protein tyrosine kinase inhibitor, selectively inhibited the growth of src or abl transformed cells in the serum-free medium resulting in about 10-fold or fivefold lower IC50 than those in the serum-containing medium. The antibiotic did not show such an effect on ras transformed cells, and the treatment of src transformed cells with other protein kinase inhibitors or cytotoxic drugs showed little IC50 shifts between the two media. Thus, this method of comparing growth inhibition in the serum-free and the serum-containing media may be useful in evaluating specific inhibitors of signaling pathways mediated by growth factors and certain oncogene products.

Tumor necrosis factor alpha-induced alteration of glycosaminoglycans in cultured vascular smooth-muscle cells.

We studied alteration of glycosaminoglycans (GAGs) induced by recombinant human tumor necrosis factor alpha (rhTNF alpha) in vascular smooth-muscle cells from bovine aorta in a culture system. It was found that rhTNF alpha at 10 ng/ml and below significantly increased the incorporation of [35S]sulfate (35S) but conversely decreased that of [3H]glucosamine (3H) into GAGs in the trypsinate fraction of the cell layer after a 24-h incubation. These results suggested that rhTNF alpha reduced the formation and/or the anchorage of sugar chains in the cell layer but enhanced their sulfation in whole GAG synthesis by the cells. In results, the ratio of 35S to 3H in the GAGs was markedly increased. This increase occurred after 24 h and longer when the cells were treated with 1.0 ng/ml rhTNF alpha. The TNF alpha-induced alteration of the incorporation of both 35S and 3H was completely blocked by anti-rhTNF alpha antibody. Other cytokines including recombinant human interleukin-1 beta and -6, and platelet-derived growth factor failed to alter the ratio of 35S to 3H in the GAGs of the trypsinate fraction of the cell layer. In cultured vascular endothelial cells from bovine aorta, however, rhTNF alpha at 1.0 ng/ml significantly decreased the incorporation of both 35S and 3H into GAGs of both the trypsinate fraction and the medium; the ratio of 35S to 3H was not changed. Characterization of GAGs in vascular smooth muscle cell trypsinate fraction revealed that rhTNF alpha at 10 ng/ml induced (i) no change of the incorporation of 3H in the hyaluronate fraction, (ii) a marked increase in the incorporation of 35S and no change of that of 3H in chondroitin sulfates (A plus C) fraction, (iii) a significant decrease in the incorporation of both 35S and 3H in the heparan sulfate fraction, and (iv) no change of the incorporation of 35S and a marked decrease in that of 3H in the dermatan sulfate fraction. In the medium, rhTNF alpha also induced various changes of GAGs. It was therefore concluded that TNF alpha may have a capacity of inducing a qualitative change of vascular smooth-muscle cell GAGs, which may be involved in the vascular pathology such as atherosclerosis.

The effect of 7 years of intense exercise training on patients with coronary artery disease.

The purpose of this study was to evaluate the effects of long-term exercise training on maximal aerobic exercise capacity, evidence of myocardial ischemia and plasma lipid-lipoprotein concentrations in patients with coronary artery disease. Nine men with coronary artery disease, aged 57 +/- 2 years, who had completed 12 months of supervised intense exercise training were restudied after 6 additional years during which they continued to exercise. The first 12 months of training resulted in a 44% increase in maximal oxygen consumption (VO2max) from 25.0 +/- 1.3 to 35.9 +/- 1.5 ml X kg-1 X min-1 (p less than 0.001). The VO2max after 6 additional years (total 7 years) of intense training was 36.8 +/- 2.4 ml X kg-1 X min-1. Plasma high density lipoprotein (HDL)-cholesterol concentration increased from 38 +/- 3 to 45 +/- 4 mg X dl-1 at 12 months and rose further to 53 +/- 5 mg X dl-1 at 6 years of follow-up (p less than 0.05). The atherogenic index (total cholesterol/HDL-cholesterol ratio) decreased from 5.8 +/- 0.4 to 4.9 +/- 0.4 by 12 months (p less than 0.01) and to 4.1 +/- 0.4 after 6 additional years of training (p less than 0.05). Although the maximal heart rate-pressure product was 14% higher after 12 months of training, maximal ST segment depression was significantly less, 0.27 +/- 0.06 versus 0.19 +/- 0.04 mV (p less than 0.05); this improvement was maintained after 6 years of additional training. These data provide evidence that the beneficial effects of a program of intense exercise training can be maintained for long periods in some motivated patients with coronary artery disease who continue to exercise.

Improvement in glucose tolerance after 1 wk of exercise in patients with mild NIDDM.

We investigated the effects of 1 wk of intense exercise on glucose tolerance in 10 men with abnormal glucose tolerance [7 had mild non-insulin-dependent diabetes mellitus (NIDDM), and 3 had impaired glucose tolerance]. The 7 days of exercise did not result in significant changes in body weight or maximal oxygen uptake. Plasma glucose concentration at 120 min averaged 227 +/- 23 mg/dl in an oral glucose tolerance test (OGTT) before and 170 +/- 18 mg/dl after the 7 days of exercise (P less than .001). There was a 36% reduction in the area under the glucose tolerance curve. Plasma insulin concentration at 120 min of the OGTT averaged 172 +/- 27 microU/ml before and 106 +/- 13 microU/ml after 7 days of exercise (P less than .001); the area under the insulin curve was decreased by 32%. In contrast to the response to 7 days of exercise, one bout of exercise did not result in an improvement in glucose tolerance. These results provide evidence that regularly performed, vigorous exercise can be effective in decreasing insulin resistance and improving glucose tolerance within 7 days in some patients with mild NIDDM.

Randomized study of orally administered fluorinated pyrimidines (capecitabine versus S-1) in women with metastatic or recurrent breast cancer: Japan Breast Cancer Research Network 05 Trial.

PURPOSE: Capecitabine and S-1 are orally administered fluorinated pyrimidines with high-level activity against metastatic breast cancer (MBC). This randomized, multicenter, phase II study compared the activities and safeties of the oral fluoropyrimidines, capecitabine and S-1, in breast cancer patients. METHODS: Patients with MBC were randomly assigned to receive capecitabine 825 g/m(2) twice daily on days 1-21 every 4 weeks or S-1 40-60 mg twice daily, according to body surface area, on days 1-28 every 6 weeks. The primary endpoint was progression-free survival (PFS). RESULTS: A total of 142 patients were enrolled and randomized to either capecitabine (N = 73) or S-1 (N = 69). Median PFS (progression-free survival) was 1.2 years for capecitabine and 1.3 years for S-1, with a hazard ratio (S-1/capecitabine) of 0.85 (95 % confidence interval [CI] 0.52-1.38) (P = 0.48 by log-rank). The confirmed objective response rates were 24.0 % for capecitabine and 23.1 % for S-1 (P = 0.938). The most common treatment-related adverse events were grade 1-2 in intensity. Thrombocytopenia (S-1: 9.2 %, capecitabine: 1.4 %; P = 0.040) and nausea (S-1: 26.2 %, capecitabine: 14.1 %; P = 0.079) were more frequent in the S-1 group, while hand-foot syndrome occurred more often in the capecitabine group (S-1: 10.8 %, capecitabine: 25.4 %; P = 0.029). CONCLUSIONS: The results of the current study demonstrate that both S-1 and capecitabine are effective and well-tolerated treatments in patients with MBC, while their adverse events were different. They are both convenient, orally administered drugs, making them attractive agents for use in outpatient treatment.

GIRADS: método de clasificación de masas anexiales, revisión sistemática y metanálisis. 10 años de experiencia / GIRADS: adnexal masses classification method, systematic review and metanalysis. 10 years of experience

OBJETIVO: evaluar la experiencia en la utilización del método GIRADS para clasificar masas anexiales a diez años de su primera publicación. MÉTODO: Se realizó búsqueda de estudios que utilizan el sistema GIRADS: Medline (Pubmed), Google Scholar y Web of Science, desde enero de 2009 hasta diciembre de 2019. Se calculó la sensibilidad y especificidad agrupada, Likelihood ratio (LR) (+) y LR (-) y Odds ratio de diagnóstico (DOR). La calidad de los estudios se evaluó con QUADAS-2. RESULTADOS: Se identificaron 15 estudios y se incluyeron 13 de ellos con 4473 masas, 878 de ellas malignas. La prevalencia media de malignidad ovárica fue del 23 % y la agrupada de 19.6%. El riesgo de sesgo fue alto en cuatro estudios para el dominio "selección de pacientes" y fue bajo en todos en todos los estudios para los dominios "prueba índice" y "prueba de referencia". La sensibilidad, especificidad, LR (+) y LR (-) agrupadas y el DOR del sistema GIRADS para clasificar las masas anexiales fueron: 96.8% (intervalo de confianza [IC] 95% = 94% - 98%), 91.2 % (IC 95 % = 85% - 94%), 11.0 (IC 95% = 6.9 -13.4) y 0.035 (IC 95% = 0.02- 0.09), y 209 (IC 95% = 99-444), respectivamente. La heterogeneidad fue alta para la sensibilidad y especificidad. De acuerdo a la metaregresión, la heterogeneidad entre los estudios se explica por la prevalencia de malignidad, múltiples observadores y la ausencia de diagnóstico histopatológico para todos los casos incluidos en un determinado estudio. CONCLUSIÓN: el sistema GIRADS tiene un buen rendimiento diagnóstico para clasificar masas anexiales.

OBJECTIVE: to evaluate the experience of using GIRADS method to classify adnexal masses ten years after its publication. METHOD: A search was carried out for studies reporting on the use of the GIRADS system in the Medline (Pubmed), Google Scholar and Web of Science databases, from January 2009 to December 2019. Pooled sensitivity and specificity, Likelihood ratio (LR) (+) and LR (-) and Diagnostic Odds ratio (DOR) were calculated. The quality of the studies was assessed by QUADAS-2. RESULTS: 15 studies were identified, and 13 of them were included with 4473 masses, of which 878 were malignant. The mean prevalence of ovarian malignancy was 23% and the prevalence pooled. of 19.6%. The risk of bias was high in four studies for the domain 'patient selection' and low for all studies for the domains 'index test' and 'reference test'. The sensitivity, specificity, pooled LR (+) and LR (-) and the DOR of the GIRADS system to classify adnexal masses were 96.8% (95% confidence interval [CI] = 94% -98%), 91.2 % (95% CI = 85% -94%), 11.0 (95% CI = 6.9-13.4) and 0.035 (95% CI = 0.02-0.09), and 209 (95% CI = 99-444), respectively. Heterogeneity was high for both sensitivity and specificity. According to meta-regression, this heterogeneity was explained by the prevalence of malignancy, the use of multiple observers, and the absence of histopathological diagnosis for all cases included in a given study. CONCLUSION: the GIRADS system has a good diagnostic performance to classify adnexal masses.

Neuronal sensitivity to opiate and opioid peptides in the bed nucleus of the stria terminalis. Effects of chronic treatment with morphine.

To characterize the phenomenon of morphine tolerance in central nervous system, the neuronal sensitivity to opiate and opioid peptides in the bed nucleus of the stria terminalis (BST) and the effects of chronic treatment with morphine were examined with thin slices of the brain of guinea pigs. Both morphine and enkephalins (D-Ala2-Met5-enkephalinamide and D-Ala2-D-Leu5-enkephalin) suppressed neuronal activity in the stria terminalis. Three days after implantation of morphine pellets the guinea pigs became tolerant to morphine and EC50 (the morphine concentration required for causing 50% of the maximal effect) was 79 microM, nearly six times the value in the control animals (EC50 = 13 microM). Cross-tolerance between morphine and enkephalins was not observed. Results of radioreceptor assay, using [3H]dihydromorphine as a ligand, showed that the binding capacity of the morphine-tolerant animals was not statistically different from that of the controls. These results suggested that tolerance to morphine cannot be explained by changes in number and/or affinity of opiate receptors, and that tolerance probably results from modification of the intracellular mechanisms mediating between the binding of morphine with the receptors and neuronal inhibition.

Phorbol ester and forskolin suppress the presynaptic inhibitory action of group-II metabotropic glutamate receptor at rat hippocampal mossy fibre synapse.

Selective activation of second messenger pathways were tested on presynaptic metabotropic glutamate receptor action at mossy fibre-CA3 synapses, using a rat hippocampal slice preparation. Application of the protein kinase C activator, phorbol 12,13-diacetate, or the adenylate cyclase activator, forskolin, markedly enhanced the mossy fibre field excitatory postsynaptic potentials, and suppressed the relative magnitude of the synaptic depression induced by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, an agonist at group-II metabotropic glutamate receptors. These effects were also observed in a low Ca2+ solution, suggesting that they were not due to saturation of transmitter release process. Inactive analogues of the respective activators (4alpha-phorbol 12,13-didecanoate and 1,9-dideoxyforskolin) neither enhanced the mossy fibre responses nor suppressed (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine-induced synaptic depression. These results suggest that the presynaptic inhibitory action of group-II metabotropic glutamate receptors at mossy fibre-CA3 synapses could be negatively regulated by protein kinase C- and cyclic AMP-dependent mechanisms.

Left ventricular dysfunction after prolonged strenuous exercise in healthy subjects.

To determine whether depressed left ventricular (LV) contractile function can occur after prolonged and strenuous exercise, 12 healthy men, 26 +/- 1 years old (mean +/- standard error of the mean) were studied. The subjects exercised on a treadmill at 69 +/- 1% of maximal O2 uptake until exhaustion (170 +/- 10 minutes). Hemodynamic variables were measured before and 10 minutes after exhausting exercise. Baseline systolic blood pressure decreased from 124 +/- 2 to 113 +/- 3 mm Hg (p less than 0.001) after exhausting exercise. LV end-diastolic diameter, measured by echocardiography, decreased from 51 +/- 1.0 to 47 +/- 1.0 mm (p less than 0.005) but LV end-systolic diameter did not change (34 +/- 1.0 vs 34 +/- 1.0 mm). Both LV fractional shortening and the mean velocity of circumferential fiber shortening decreased (33 +/- 1 vs 28 +/- 1%; p less than 0.01 and 1.09 +/- 0.4 vs 0.97 +/- 0.05 circ/s; p less than 0.025) despite a lower end-systolic wall stress (sigma es = 88 +/- 4 vs 82 +/- 5, X 10(3) dynes/cm2; p less than 0.05) after prolonged exhausting exercise. A repeat bout of exercise of the same intensity but brief in duration (10 minutes) resulted in increases in LV fractional shortening (p less than 0.001) and mean velocity of circumferential fiber shortening (p less than 0.001), and a decrease in LV end-diastolic diameter (50 +/- 1.0 to 48 +/- 1.0 mm; p less than 0.05) at heart rates comparable to those attained after prolonged exhausting exercise. The results suggest that prolonged strenuous exercise may result in impaired LV function in healthy young subjects.

Programmed filtration, a new method for removing large molecules and regulating albumin leakage during hemodiafiltration treatment.

During hemodiafiltration (HDF) treatment for chronic renal failure patients, replacing large volumes using high-flux membranes with relatively large pores is preferred from the standpoint of enhancing the elimination of large molecules (10 to 50 kd). Aggressive protein-permeable treatment often results in massive leakage of essential albumin, however, which may cause fatigue, hypotension, and a decrease in the plasma albumin concentration in some patients. During 5-hour conventional HDF treatment with the filtration rate or pressure set at constant values, fractional albumin loss in the dialysate was assayed, which revealed that the albumin concentration in the dialysate showed a maximum value in the beginning with a steep decline within 1 hour. Approximately 40% to 50% of the total amount of albumin leakage occurred during the first 30 minutes. Concomitantly the large molecules transferred into the pores by aggressive filtration during the beginning partially plugged the pores, resulting in a decrease in the permeability for beta(2)-microglobulin. From the standpoint of achieving the highest clearance for large molecules, while suppressing albumin leakage below the acceptable range, the optimal profiles for filtration conditions in HDF have been proposed, in which either the transmembrane pressure is regulated according to the sigmoid curve in the pressure control manner or the flow rate is set along the concave in the flow control manner. The profiles of pressure or flow as a function of time have been programmed and installed in a HDF machine to perform an optimal HDF treatment automatically. The new filtration methods gave significantly higher beta(2)-microglobulin removal and lower albumin leakage than conventional HDF methods with constant filtration.

Specific inhibition of cytoplasmic protein tyrosine kinases by herbimycin A in vitro.

Herbimycin A is an antibiotic which reverses transformation caused by various src related oncogenes. The reversion of transformation is restricted to cells transformed by tyrosine kinase coding oncogenes, and accompanies a decrease in kinase activity of the oncogene products. We have shown in vitro that herbimycin A directly inactivates p60v-src kinase by conjugating with SH group(s) of the kinase, raising the possibility that the molecular target of the antibiotic for reversion of v-src transformation is the p60v-src itself. To investigate the relevance of its in vitro tyrosine kinase inactivating activity to in vivo transformation reversing activity, we examined the specificity of herbimycin A for inhibition of cAMP-dependent kinase, protein kinase C and p210bcr-abl tyrosine kinase in vitro. Herbimycin A had no inhibitory effect on the activities of cAMP-dependent kinase or protein kinase C, whereas the SH-reagent N-(9-acridinyl)maleimide, which inactivates p60v-crc in vitro by a mechanism similar to that of herbimycin A, blocked the two serine/threonine kinases. On the other hand, the activity of p210bcr-abl tyrosine kinase was inhibited by herbimycin A treatment. The results indicate that herbimycin A specifically binds to reactive SH group(s) of cytoplasmic protein tyrosine kinases, and confer the biochemical basis for its selectivity in reversing cell transformation.