Beneficial effects of pentoxifylline in patients with idiopathic dilated cardiomyopathy treated with angiotensin-converting enzyme inhibitors and carvedilol: results of a randomized study.

BACKGROUND: We previously reported beneficial effects of pentoxifylline, a xanthine-derived agent known to inhibit the production of tumor necrosis factor-alpha, in patients with idiopathic dilated cardiomyopathy treated with diuretics, digoxin, and ACE inhibitors. Since then, 3 large clinical trials showed important clinical benefits of beta-blockers in this population. Therefore, we designed the present study to establish whether in patients with heart failure already receiving treatment with ACE inhibitors and beta-blockers, the addition of pentoxifylline would have an additive beneficial effect. METHODS AND RESULTS: In a single-center, prospective, double-blind, randomized, placebo-controlled study, 39 patients with idiopathic dilated cardiomyopathy were randomized to pentoxifylline 400 mg TID (n=20) or placebo (n=19) if they had a left ventricular ejection fraction <40% after 3 months of therapy with digoxin, ACE inhibitors, and carvedilol. Primary end points were New York Heart Association functional class, exercise tolerance, and left ventricular function. Patients were followed up for 6 months. Five patients died (3 in the placebo group). Patients treated with pentoxifylline had a significant improvement in functional class compared with the placebo group (P:=0.01), with an increment in exercise time from 9.5+/-5 to 12.3+/-6 minutes (P:=0.1). Left ventricular ejection fraction improved from 24+/-9% to 31+/-13%, P:=0.03, in the treatment group. CONCLUSIONS: In patients with idiopathic dilated cardiomyopathy, the addition of pentoxifylline to treatment with digoxin, ACE inhibitors, and carvedilol is associated with a significant improvement in symptoms and left ventricular function.

Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping.

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.

Peripartum cardiomyopathy: analysis of clinical outcome, left ventricular function, plasma levels of cytokines and Fas/APO-1.

OBJECTIVES: 1) To evaluate the outcome of patients with peripartum cardiomyopathy (PPC) on current treatment for heart failure, 2) to assess the circulating plasma levels of cytokines and Fas receptors and 3) to identify predictors of prognosis. BACKGROUND: Previous studies in patients with PPC were done when angiotensin-converting enzyme (ACE) inhibitors and beta-adrenergic blocking agents were not routinely used in heart failure. Inflammatory cytokines play an important role in the pathogenesis and progression of heart failure of other etiologies. However, there is a paucity of data regarding cytokine expression in patients with PPC. Plasma concentrations of Fas receptors (an apoptosis-signalling receptor) have not been reported in this population. METHODS: We followed prospectively 29 consecutive black women with PPC. All patients were treated with diuretics, digoxin, enalapril and carvedilol. Echocardiograms were performed at baseline and after six months of treatment. Cytokine and soluble Fas/APO-1 plasma levels were measured at baseline. RESULTS: Tumor necrosis factor-alpha, interleukin-6 and Fas/APO-1 levels were significantly elevated in the study patients compared with 20 healthy volunteers. Eight patients died. sFas/APO-1 levels were significantly higher in patients who died compared with survivors (8.98 +/- 4.5 vs. 5.33 +/- 3 U/ml, respectively, p = 0.02). At six months, ejection fraction improved from 26.7 +/- 10 to 42.7 +/- 16%, p = 0.00003, with an increment of more than 10 U in 10 patients (28.1 +/- 4 to 51.9 +/- 8%, p = 0.000008). CONCLUSIONS: Cytokine and sFas levels are elevated in patients with PPC. Despite treatment with ACE inhibitors and beta-blockers, mortality remains high. However, in 34% of the patients, left ventricular function almost completely normalized.

Ephrin-B3, a ligand for the receptor EphB3, expressed at the midline of the developing neural tube.

The ephrins are a family of ligands that bind to Eph family receptor tyrosine kinases, and have been implicated in axon guidance and other patterning processes during vertebrate development. We describe here the identification and characterization of murine ephrin-B3. The cDNA encodes a 340 amino acid transmembrane molecule, most closely related to the two other known transmembrane ligands, ephrin-B1 and ephrin-B2. In addition to homology in their extracellular receptor binding domains, these transmembrane ligands share striking homology between their cytoplasmic domains, with 31 of the last 34 amino acids of ephrin-B3 being identical to ephrin-B2, suggesting functional interactions of the cytoplasmic tail. While most Eph family ligands are promiscuous in their interactions with Eph receptors, binding studies with the five receptors known to bind other transmembrane ligands only revealed a high affinity interaction of ephrin-B3 with EphB3, with a dissociation constant of approximately 1 nM. In situ hybridization of mouse embryos showed ephrin-B3 is expressed prominently at the dorsal and ventral midline of the neural tube, particularly in the floor plate, a structure with key functions in patterning the nervous system. The isolation of this ligand may help to elucidate the molecular basis of patterning activities at the neural tube midline.

Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.

Receptor protein tyrosine kinases (RTKs) transmit downstream signals via interactions with secondary signaling molecules containing SH2 domains. Although many SH2-phosphotyrosyl interactions have been defined in vitro, little is known about the physiological significance of specific RTK/SH2 interactions in vivo. Also, little is known about the mechanisms by which specific RTKs interact with and/or are regulated by specific protein tyrosine phosphatases (PTPs). To address such issue, we carried out a genetic analysis of the previously reported biochemical interaction between the RTK c-Kit, encoded at the W locus, and the SH2-containing non-transmembrane PTP SHP1, encoded at the motheaten (me) locus (1). Mice carrying a kinase-defective allele of c-Kit (Wv/+) were crossed with me/+ mice, which carry one effectively null allele of SHP1, and then backcrossed to generate all possible allelic combinations. Our results indicate strong intergenic complementation between these loci in hematopoietic progenitor cells. Compared to progenitors purified from normal mice, bone marrow progenitor cells (lin-) from me/me mice markedly hyper-proliferated in response to Kit ligand (KL). stimulation. Superimposition of the me/me genotype increased the number of one marrow-derived CFU-E from Wv/+ mice. Conversely, the presence of one or two copies of Wv decreased the number of macrophages and granulocytes in me/me lung, skin, peripheral blood and bone marrow, thereby decreasing the severity of the me/me phenotype. The decrease in dermal mast cells in Wv/Wv mice was rescued to levels found in Wv/+mice by superimposition of the me/me genotype. Surprisingly, however, the presence or absence of SHP1 had no effect on the proliferative response of bone marrow-derived cultured mast cells to KL or IL3 ex vivo. Nevertheless, the immediate-early response to KL stimulation, as measured by KL-induced tyrosyl phosphorylation, was substantially increased in mast cells from Wv/+:me/me compared to Wv/ +:+/+ mice, strongly suggesting that SHP1 directly dephosphorylates and regulates c-Kit. Taken together, our results establish that SHP1 negatively regulates signaling from c-Kit in vivo, but in a cell type-specific manner.

The spectrum of meningitis in a population with high prevalence of HIV disease.

We studied the spectrum of meningitis and impact of HIV infection retrospectively (8 months) and prospectively (4 months) in 284 adult patients with meningitis hospitalized in Soweto, South Africa. Tuberculous meningitis (TBM) was the most common cause of meningitis (25.4%) followed by acute bacterial meningitis (ABM; 22.5%), acute viral meningitis (14.1%) and cryptococcal meningitis (13%). The in-hospital mortality was > 40% in TBM, ABM, cryptococcal meningitis, the neurosurgery and the parameningeal/parenchymal groups. At least 37.3% of all patients were HIV-seropositive (only 67.9% of patients were tested). In at least 27% of the study group the meningitis was an AIDS-defining illness (TBM, cryptococcal meningitis). Only 56.2% of patients with ABM had positive cultures (CSF or blood), of which Streptococcus pneumoniae was by far the most frequently found organism (35.8%). The spectrum of meningitis in HIV-affected communities in Africa can be expected to change towards a predominance of TBM and cryptococcal meningitis.

PIP: To evaluate the spectrum of meningitis and its impact on human immunodeficiency virus (HIV) infection, 284 adults hospitalized with meningitis in Soweto, South Africa, were studied. Tuberculosis meningitis (TBM) was the most common cause of meningitis (25.4%), followed by acute bacterial meningitis (ABM; 22.5%), acute viral meningitis (AVM; 14.1%), and cryptococcal meningitis (13%). The in-hospital mortality rate exceeded 40% in TBM, ABM, cryptococcal meningitis, the neurosurgery group, and the parameningeal/parenchymal group. Only 56.2% of patients with ABM had positive blood or cerebrospinal fluid cultures. 37.3% of the 193 patients tested for HIV were seropositive. All patients with cryptococcal meningitis and at least 54% of those with TBM were HIV-infected. Moreover, at least 27% of the study population presented with an acquired immunodeficiency syndrome (AIDS)-defining illness such as cryptococcal meningitis or TBM. The high mortality rates observed among meningitis patients in this series reflect immunosuppression associated with HIV infection or malnutrition, late presentation at a hospital, lack of access to medical care, and failure on the part of some primary care providers to consider a diagnosis of meningitis. Underlying HIV infection in increasing numbers of meningitis patients can be expected to produce a need for more hospital beds and increased medical expenditures in South Africa.

Cystic lung disease in systemic sclerosis: a case report with high resolution computed tomography findings.

A 31-year old black female presented with limited scleroderma. Although she had no chest symptoms, chest radiographs showed a basal reticular pattern with large cysts in both lung fields. High resolution computed tomography revealed almost complete effacement of normal lung architecture by multiple large cysts, honeycombing and interstitial fibrosis. Cystic lung disease is an uncommon manifestation of interstitial lung disease in systemic sclerosis, and its natural history needs to be better defined.

Similarities and differences in the way transmembrane-type ligands interact with the Elk subclass of Eph receptors.

The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signaling events taking place, we have undertaken a comparative analysis of ligand-receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellular transformation assays, we find that the transmembrane-type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The common receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, specifically, but weakly, binds Cek10 and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand-receptor interactions as presented in this study will be important for the design and interpretation of in vivo experiments, e.g., loss-of-function studies in transgenic mice.

ELF-2, a new member of the Eph ligand family, is segmentally expressed in mouse embryos in the region of the hindbrain and newly forming somites.

The Eph receptors are the largest known family of receptor tyrosine kinases and are notable for distinctive expression patterns in the nervous system and in early vertebrate development. However, all were identified as orphan receptors, and only recently have there been descriptions of a corresponding family of ligands. We describe here a new member of the Eph ligand family, designated ELF-2 (Eph ligand family 2). The cDNA sequence for mouse ELF-2 indicates that it is a transmembrane ligand. It shows closest homology to the other known transmembrane ligand in the family, ELK-L/LERK-2/Cek5-L, with 57% identity in the extracellular domain. There is also striking homology in the cytoplasmic domain, including complete identity of the last 33 amino acids, suggesting intracellular interactions. On cell surfaces, and in a cell-free system, ELF-2 binds to three closely related Eph family receptors, Elk, Cek10 (apparent ortholog of Sek-4 and HEK2), and Cek5 (apparent ortholog of Nuk/Sek-3), all with dissociation constants of approximately 1 nM. In situ hybridization of mouse embryos shows ELF-2 RNA expression in a segmental pattern in the hindbrain region and the segmenting mesoderm. Comparable patterns have been described for Eph family receptors, including Sek-4 and Nuk/Sek-3, suggesting roles for ELF-2 in patterning these regions of the embryo.

Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map.

Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well-studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. We recently cloned ELF-1 as a ligand for Eph family receptors. Here, RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. Binding activity detected with alkaline phosphatase fusions of ELF-1 and Mek4 also reveals gradients and provides direct evidence for molecular complementarity of gradients in reciprocal fields. ELF-1 and Mek4 may therefore play roles in retinotectal development and have properties predicted of topographic mapping labels.

Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases.

The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. Recently, a small family of ligands, structurally related to the B61 protein, was identified. Binding of these ligands to Eph-related receptors did not, however, elicit measurable biological signals in cultured cells. In order to study functional interactions between B61-related ligands and Eph-related receptors, we constructed chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor. Expression and activation of such chimeric receptors in NIH 3T3 cells induced transformation in focus formation assays. Membrane-bound LERK2 ligand is shown to signal through three different Eph-related receptors, namely Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling.

Conservation in human and mouse Pur alpha of a motif common to several proteins involved in initiation of DNA replication.

Pur alpha is a sequence-specific single-stranded DNA-binding protein with affinity for an element present in several eukaryotic origins of DNA replication (ori) and gene regulatory regions. We report here the cDNA sequence for mouse pur alpha and an extraordinary degree of conservation between human and mouse Pur alpha (hPurA and mPurA, respectively). There are only two single-amino-acid (aa) changes between hPurA (322 aa) and mPurA (321 aa). One PurA region of 22 aa, termed the 'psycho' motif, possesses significant homology to a counterpart in the SV40 large T-antigen, to several other transforming proteins of DNA tumor viruses, and to certain cellular proteins in yeast and human cells that may also be involved in the initiation of DNA replication.

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein.

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

A major site of DNA bending is located 1.6 kb upstream of the P1 transcription start site of the human c-myc gene, near the center of a reported zone of initiation of DNA replication. A repeated, purine-rich element, termed PUR, at the bend site is specifically bound by a protein in HeLa cell nuclear extracts. This protein has specific affinity for the purine-rich single strand of the element. Methylation interference maps a pattern of specific contact points with guanosine bases in a 24-mer oligonucleotide containing the element. UV cross-linking reveals that contact is made by a polypeptide of approximately 28 kDa. The PUR element is present at origins of replication and in gene flanking regions in a variety of eukaryotes from yeasts through humans. The consensus sequence GGNNGAGGGAGARRRR has been derived. This element is present near centers of regions of two mammalian loci (human c-myc and hamster dhfr) recently reported as initiation zones for DNA replication. A 24-mer oligonucleotide representing the hamster dhfr version of the PUR element effectively competes with the human c-myc version for binding to Pur.

Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194.

The complete nucleotide sequence of pADB201, a 1.7-kilobase cryptic plasmid from Mycoplasma mycoides subsp. mycoides, is reported. The sequence contains a single large open reading frame capable of coding for a polypeptide of up to 198 codons long. The sequence of the putative polypeptide shows significant similarity to that of the repF gene product of staphylococcal plasmid pE194.

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes.

The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.

Isolation and restriction endonuclease analysis of a mycoplasma plasmid.

A 1.7-kb plasmid was isolated from a large colony type strain of Mycoplasma mycoides subsp. mycoides. It was cloned into the Escherichia coli vector M13mp19, and DNA from the resultant recombinant was used to construct a restriction map of the plasmid. Because mycoplasmas show deviation from normal coding patterns, the availability of a mycoplasma plasmid could be useful in the development of a cloning vector for them.