Expert proposal to analyze the combination of aortic and mitral regurgitation in multiple valvular heart disease by comprehensive echocardiography.

The assessment of valvular pathologies in multiple valvular heart disease by echocardiography remains challenging. Data on echocardiographic assessment-especially in patients with combined aortic and mitral regurgitation-are rare in the literature. The proposed integrative approach using semi-quantitative parameters to grade the severity of regurgitation often yields inconsistent findings and results in misinterpretation. Therefore, this proposal aims to focus on a practical systematic echocardiographic analysis to understand the pathophysiology and hemodynamics in patients with combined aortic and mitral regurgitation. The quantitative approach of grading the regurgitant severity of each compound might be helpful in elucidating the scenario in combined aortic and mitral regurgitation. To this end, both the individual regurgitant fraction of each valve and the total regurgitant fraction of both valves must be determined. This work also outlines the methodological issues and limitations of the quantitative approach by echocardiography. Finally, we present a proposal that enables verifiable assessment of regurgitant fractions. The overall interpretation of echocardiographic results includes the symptomatology of patients with combined aortic and mitral regurgitation and the individual treatment options with respect to their individual risk. In summary, a reproducible, verifiable, and transparent in-depth echocardiographic investigation might ensure consistent hemodynamic plausibility of the quantitative results in patients with combined aortic and mitral regurgitation.

Expert proposal to characterize cardiac diseases with normal or preserved left ventricular ejection fraction and symptoms of heart failure by comprehensive echocardiography.

Currently, the term "heart failure with preserved left ventricular ejection fraction (HFpEF)" is based on echocardiographic parameters and clinical symptoms combined with elevated or normal levels of natriuretic peptides. Thus, "HFpEF" as a diagnosis subsumes multiple pathophysiological entities making a uniform management plan for "HFpEF" impossible. Therefore, a more specific characterization of the underlying cardiac pathologies in patients with preserved ejection fraction and symptoms of heart failure is mandatory. The present proposal seeks to offer practical support by a standardized echocardiographic workflow to characterize specific diagnostic entities associated with "HFpEF". It focuses on morphological and functional cardiac phenotypes characterized by echocardiography in patients with normal or preserved left ventricular ejection fraction (LVEF). The proposal discusses methodological issues to clarify why and when echocardiography is helpful to improve the diagnosis. Thus, the proposal addresses a systematic echocardiographic approach using a feasible algorithm with weighting criteria for interpretation of echocardiographic parameters related to patients with preserved ejection fraction and symptoms of heart failure. The authors consciously do not use the diagnosis "HFpEF" to avoid misunderstandings. Central illustration: Scheme illustrating the characteristic echocardiographic phenotypes and their combinations in patients with "HFpEF" symptoms with respect to the respective cardiac pathology and pathophysiology as well as the underlying typical disease.

Agarose gel electrophoretic evidence for domains of nuclear DNA linked with bonds cleavable with sulfhydryl molecules.

Complexes of intact nuclear DNA with proteins undissociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage lambda DNA) into smaller-Mr forms. Following limited restriction endonuclease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60-110 kbp range. These 60-110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.

Differential c-myc protein expression in Burkitt's lymphomas and EBV-transformed lymphoblastoid lines.

The levels of c-myc protein expression in three types of Epstein-Barr virus (EBV) transformed human B-cell derived lines were examined with an ELISA assay. Six independently maintained sublines of the same EBV-transformed pro-B-cell line (FLEB-14), six B-cell lines (LCL) and six Burkitt's lymphoma lines (BL) were compared. The average amount of c-myc protein, calculated from at least three independent tests on each line, differed between the three groups. Expressed in relative units, the ratio of the means was 1:2:5 for the LCL:FLEB:BL lines. The differences were statistically significant at P less than 0.01.

Unexpected bone marrow uptake of thallium-201 in nonsecretory myeloma.

A 43-yr-old man was referred for any possible parathyroid abnormality that could explain his hypercalcemia and slightly increased parathormone levels. The thallium-technetium scan showed a diffuse abnormal thallium uptake incidentally in the bone marrow, otherwise parathyroid scan appearance was normal. He had an essentially normal bone scan, although subsequent nanocolloid scintigraphy demonstrated bone marrow expansion. Further investigations, including a bone marrow aspiration biopsy, confirmed the diagnosis of nonsecretory myeloma. This finding suggests that 201Tl imaging can be a useful tool to investigate those patients suffering from similar myeloid disorders causing bone marrow hyperplasia.

Clinical comparison of technetium-99m-EC, technetium-99m-MAG3 and iodine-131-OIH in renal disorders.

UNLABELLED: Technetium-99m-ethylenedicysteine has recently been developed for renal function studies. The pharmacokinetics of 99mTc-EC were studied by constant infusion technique and compared with 99mTc-MAG3 and 131I-OIH in 11 patients with various renal disorders. METHODS: After giving a 7.4 MBq 131I-OIH and 90-110 MBq 99mTc-EC or 99mTc-MAG3 bolus, a constant infusion (1MBq/ml 99mTc-agent and 0.07 MBq/m 131I-OIH was started. Sixteen blood and five urine samples were obtained over three hr. RESULTS: The renal clearance of 99mTc-EC was higher than that of 99mTc-MAG3. The 99mTc-EC/OIH and 99mTc-MAG3/OIH ratios were 0.75 +/- 0.05 and 0.55 +/- 0.10 (p = 0.00087), respectively. The distribution volume of 99mTc-EC was also higher than that of 99mTc-MAG3 (15722 +/- 4644 and 9509 +/- 2788 ml/1.73m2, respectively; p = 0.072). The 99mTc-EC/OIH and 99mTc-MAG3/OIH distribution volume ratios were 1.03 +/- 0.14 and 0.55 +/- 0.10, respectively (p = 0.0003). The 60-min excretion values of 99mTc-EC and 99mTc-MAG3 were compared to that of OIH. The 99mTc-EC/OIH and 99mTc-MAG3/OIH excretion ratios were 0.96 +/- 0.06 and 1.07 +/- 0.10, respectively (p = 0.162). The protein binding of 99mTc-EC and OIH were found to be 34% +/- 4 and 66% +/- 5, respectively (p < 0.0001). The red cell binding of 99mTc-EC was negligible (3% +/- 1.2) in comparison to OIH (27% +/- 3; p < 0.0001). CONCLUSION: This limited study demonstrates the pharmacokinetic and renal clearance properties of 99mTc-EC. This agent has good potential for renal function evaluation.

Influence of transforming growth factor-beta (TGF-beta) on the immunoglobulin production by EBV-infected B cell cultures.

TGF-beta inhibits the proliferation of human B lymphocytes stimulated by a variety of activators, including EBV. However, EBV-immortalised cells are refractory to TGF-beta. The influence of TGF-beta on B cell maturation varies, apparently depending on the origin of the B lymphocytes and their maturation/activation state, the strength of the stimulus and the presence of cofactors. We investigated the effect of TGF-beta on immunoglobulin production by 5-day-old EBV-infected B cells. TGF-beta added at the initiation of the cultures inhibited IgM, IgG and IgA secretion by decreasing the numbers of secretory cells. The inhibition of IgM secretion was strongest. At the cytoplasmic level, TGF-beta reduced the expression of IgM heavy, lambda and kappa light chains but not IgG and IgA heavy chains. However, the IgM production by an established EBV-transformed B cell line was not affected by TGF-beta. Thus, TGF-beta inhibited EBV-induced maturation of the B cells until they acquired a transformed state. We discuss the relevance of these findings for the potential role of TGF-beta on EBV infection.

Cerebellar and subcortical blood flow abnormalities in children with partial epilepsy.

Cerebellar and cerebral subcortical blood flow in 41 children with partial epilepsy and 6 normal controls was investigated during the interictal state using single photon emission computed tomography with technetium-99m hexamethylpropyleneamineoxime. Seventeen of 41 patients had been treated with antiepileptic drugs (AEDs) for 4.65 +/- 3.80 years (range 0.2-12) and 24 patients were drug-free. Unilateral hypoperfusion of cerebellum and cerebral subcortical gray matter was demonstrated in 11 (28%) and 16 (40%) patients, respectively. Most of them also had focal cerebral cortical perfusion abnormalities, ipsilateral or contralateral to the cerebellar and cerebral subcortical hypoperfusion. The mean asymmetry indices of cerebellar blood flow in the medicated and the unmedicated patients were significantly higher than in the control cases (P < 0.02 and P < 0.04), whereas the differences in the asymmetry indices in cerebral subcortical areas were insignificant. AED therapy did not affect the perfusion of cerebellum and cerebral subcortical regions (P > 0.05 and P > 0.05). Our results suggest that functional alterations on anatomically connected remote areas in patients with partial epilepsy are not related to the drug effect and probably due to primary epileptogenic activity.

Cell phenotype-dependent splicing reflecting differential promoter usage for EBNA transcripts in EBV-carrying cells.

Three types of virus-host cell interactions have been described in cells latently infected with EBV: EBNA 1 expression in type I Burkitt's lymphoma cell lines (BL), EBNA 1, LMP1 and 2 expression in most nasopharyngeal carcinomas (NPC) and EBNA 1-6 with LMP 1 and 2 expression in group III BL-lines as well as lymphoblastoid cell lines (LCL). Two group I BL lines that express only EBNA 1 were found to initiate their EBNA 1 mRNA transcription from a promoter in the Bam HI Q-fragment. They use a sequence at +210 bp relative to the Fp transcription initiation site in group I BL cell lines. The Fp promoter-region seems to be activated in the lytic cycle. LCLs initiate their transcription from one of several upstream sites, usually the Cp promoter or, less frequently, one of several Wp-promoters. Using RNA-reverse transcription polymerase chain reaction (RT-PCR), we have now shown that EBV carrying cells that do not express EBNA 2-6 always splice their EBNA mRNA at the Q-exon, while EBNA 2-6 positive cells use either the Cp or one of the Wp promotors. When EBNA 2-6 are downregulated by somatic cell hybridisation between EBNA 1-6 positive B-cell lines and non B-cells of hematopoetic, epithelial or fibroblastic origin that express the phenotype of the non-B cell parent, the parental usage of Cp/Wp is switched off, and the Q-exon is activated. NPC cells show the same pattern of promoter usage as the hybrids with non-B phenotype. Group III BL cells use both promoter regions. Thus, the virus can use two alternative programs, depending on the cell phenotype. The "EBNA-1 only" program is activated from the Q-promoter. In cells with an immunoblastic (LCL or BL group III) phenotype, the upstream Cp/Wp promoters generate a 100 kb. long pre-mRNA, from which all the EBNAs are spliced. As a rule, only one of the two programs is used for each phenotype, except for the BL group III cells that began as group I but subsequently developed into a more LCL-like cell. Such cells used both promoter regions, with or without activation of the lytic cycle.

Functional interaction of Oct transcription factors with the family of repeats in Epstein-Barr virus oriP.

The family of repeats (FR) is a major upstream enhancer of the Epstein-Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.

The role of Epstein-Barr virus in lymphomas of HIV-carriers.

A substantial proportion of the lymphomas in HIV-carriers are EBV positive. Together with the fact that there are multiple signs of EBV-activation in AIDS-patients and patients with ARC or PGL, this suggests that these virus carrying tumors develop as results of the immunosuppression, and that EBV has an important role in their pathogenesis.

Analysis of methylation patterns in the regulatory region of the latent Epstein-Barr virus promoter BCR2 by automated fluorescent genomic sequencing.

We analyzed the methylation patterns of CpG dinucleotides in the regulatory region of the latent Epstein-Barrvirus (EBV) promoter BCR2 (also called C promoter, Cp) using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. BCR2 is one of the alternative promoters for transcripts encoding the growth-transformation-associated nuclear antigens EBNA 1-6 which are expressed in a host cell phenotype dependent manner. Well characterized clones isolated from the Burkitt's lymphoma (BL) line Mutu differing from each other as to their phenotype and EBV latent gene expression were used in the present study. We found that in Mutu BL III clone 99 which is actively using the BCR2 promoter the regulatory sequences are unmethylated with two exceptions (position 10702 and 10799). In contrast, there are 15 methylated cytosines in the same region in Mutu BL I clone 216 where the BCR2 promoter is silent. Cytosines which are potential targets of DNA methyltransferase in the immediate vicinity or within the attachment sites of cellular C promoter binding factors CBF1 and CBF2 remained hypomethylated in Mutu BL I clone 216. This suggests a role for a hypermethylated region (nucleotides 10666 -10865, -639 to -440 bases upstream from the beginning of the TATA box at position 11305) in silencing of the BCR2 promoter in these cells.

Effect of transforming growth factor-beta 1 and -beta 2 on the proliferation of Burkitt lymphoma and lymphoblastoid cell lines.

We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.

Host-cell-phenotype-dependent control of the BCR2/BWR1 promoter complex regulates the expression of Epstein-Barr virus nuclear antigens 2-6.

Epstein-Barr virus nuclear antigens (EBNAs) are expressed in a cell-phenotype-dependent manner. EBNA 1 is regularly expressed in all Epstein-Barr virus-carrying cells, whereas EBNAs 2-6 are only expressed in Epstein-Barr virus-carrying cells with a lymphoblastoid phenotype including group III Burkitt lymphoma (BL) lines positive for B-cell activation markers. Transcripts are initiated at the BCR2 or exceptionally at one BWR1 promoter in lymphoblastoid cell lines and group III BL lines. In group I BL lines, nasopharyngeal carcinoma, and the somatic cell hybrids, where EBNAs 2-6 are downregulated, the BCR2/BWR1 promoter complex is inactive or switched off. Upregulation of EBNAs 2-6 in group III BL cells and in 5-azacytidine-treated group I BL cells accompanies the activation of the silent BCR2/BWR1 promoters. Activation of BCR2 parallels demethylation of at least one CpG pair in the same promoter region. The activity of BCR2/BWR1 promoter complex depends on a particular B-cell phenotype. EBNA 1 transcription must be initiated at another promoter in cells that express only EBNA 1.

Maternal cocaine abuse resulting in necrotizing enterocolitis. An experimental study in a rat model. II. Results of perfusion studies.

During the last decade, several publications have appeared associating the maternal use of cocaine and subsequent development of necrotizing enterocolitis (NEC). In 1994, the effects of cocaine in pregnant rats had been reported by this group: a significant decrease in the number of live births, mean birth weight and mean placental weight. In addition, histopathologic examinations revealed severe inflammation and degenerative vascular changes in the uterus and placenta. Severe histopathologic changes resembling NEC such as focal necrosis, necrobiosis, and hemorrhagic inflammatory changes in the gastrointestinal tract of the embryos were also reported. The aim of the second part of this study was to assess the hemodynamic effects of cocaine HCI in pregnant rats and the results of perfusion studies in the uterus, placenta, and fetuses to determine a relation between the dose of drug, hemodynamic changes, and degree of histopathologic findings. Forty-seven Wistar albino rats and 91 rat fetuses were studied: group A (pregnant rats), 16 rats and 91 rat fetuses, group B (nonpregnant rats), 31 rats. Each group was divided into subgroups of cocaine-abused and non-cocaine-abused rats. In each group 2-3 mCi technetium Tc-99m methoxyisobutyl-isonitryl (Sesta MIBI) was injected into the tail vein. Radioactivity counts per g tissue (cps/g) in the uterus, placenta, and fetus were assessed by gamma counter. Cocaine 75 mg/kg per day severely decreased the perfusion of the uterus, placenta, and fetuses. These impairments were statistically significant. In lower doses (30-50 mg/kg per day) no statistically significant changes were observed in the perfusion of the uterus and placenta, but a significant decrease in fetal perfusion was seen. In group B, no significant changes in the perfusion of the uterus due to cocaine were seen. Thus, maternal cocaine abuse results in a reduction in perfusion of the uterus, placenta, and fetus. There was a dose-dependent correlation between the perfusion changes and the development of NEC-like histopathologic changes: the higher the cocaine dose received by the mother, the higher the level of placental and fetal injury. We suggest that perinatal cocaine exposure should be considered a high risk for development of NEC in rat fetuses and embryos. For this reason, infants with a history of possible maternal cocaine abuse or positive urinary cocaine metabolites have to be followed very carefully for NEC.

B cell phenotype-dependent expression of the Epstein-Barr virus nuclear antigens EBNA-2 to EBNA-6: studies with somatic cell hybrids.

The expression of the transformation-associated Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) 1 to 6 and membrane protein LMP-1 was studied in a series of somatic cell hybrids derived from the fusion of the EBV-transformed lymphoblastoid cell line (LCL) KR-4, and the EBV-carrying Burkitt's lymphoma lines Daudi, P3HR-1 and Raji, with non-B cell lines of fibroblast, erythroid, myeloid and epithelial origin. Expression of EBNAs 2 to 6 was down-regulated in the hybrids in parallel with extinction of the B cell markers CD19, CD20, CD21, CD23, HLA class II, and surface or cytoplasmic immunoglobulin. LMP-1 was expressed independently of EBNA-2 in hybrids derived by the fusion of the LMP-1-positive KR-4 and P3HR-1 cell lines with epithelial and myeloid cells, respectively. LMP-1 was down-regulated in hybrids derived by the fusion of P3HR-1 with an erythroid cell line and in the hybrid between Raji and a mouse fibrosarcoma line. EBNA-1 was the only EBV antigen that was regularly expressed in the hybrids regardless of the dominating cellular phenotype. The autonomous expression of EBNA-1 suggests that its regulatory pathway is independent of phenotype-associated cellular or viral factors. In contrast, the expression of EBNAs 2 to 6 appears to require a B cell environment. EBNA-2 was shown to contribute to the regulation of LMP expression in B cells. We show that in LCL-carcinoma hybrids the dominating epithelial phenotype is permissive for LMP expression in the absence of EBNA-2.

Epstein-Barr virus-transformed pro-B cells are prone to illegitimate recombination between the switch region of the mu chain gene and other chromosomes.

Six independently maintained sublines of FLEB 14, a fetal-liver-derived Epstein-Barr virus-transformed pro-B cell line that has not yet rearranged its immunoglobulin genes, were examined after in vitro propagation during 19-36 months. Two lines showed no immunoglobulin heavy chain gene rearrangement, whereas one allele was rearranged with breakpoints inside the switch region of the mu chain gene in the remaining four. These rearrangements had been generated by the translocation of different chromosome fragments to the immunoglobulin heavy chain gene cluster-carrying 14q32 band in each of the four lines. Previously, a similar rearrangement was found in a fifth subline concurrently with a reciprocal 6;14 translocation. The transposed pieces have been derived from chromosomes 16 and 18 in two of the more recently rearranged lines. Their origins could not be determined in the remaining two lines, but they were different from each other and the other three 14q+ markers. The 14q+ marker-carrying variant has replaced its diploid progenitor suggesting that the translocation has conveyed some in vitro growth advantage on its carrier. This was also supported by the duplication of the 14q+ marker and the loss of its normal chromosome 14 homologue in one subline during serial culturing. The vulnerability of the switch region of the mu chain gene to illegitimate recombination at the pro-B stage and the possible relevance of this finding for the origin of the Burkitt lymphoma-associated 8;14 (immunoglobulin heavy chain gene cluster/MYC) translocation is discussed.