Glycoprotein B of bovine herpesvirus type 4: Its phylogenetic relationship to gB equivalents of the herpesviruses

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the -herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.     

Evaluation of BBL crystal MRSA ID system.

The BBL Crystal system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) was evaluated for its accuracy in identifying oxacillin resistance in Staphylococcus aureus by testing of mec-specific-gene-positive and -negative isolates. Although the manufacturer makes no claim for use of the product for testing of staphylococci other than S. aureus, the product's potential utility in detecting oxacillin resistance in isolates of mec gene-positive and -negative Staphylococcus epidermidis was also explored. All mec gene-negative staphylococci yielded a negative MRSA ID test reaction. There was a close correlation between mec gene positivity and a positive reaction in the methicillin-resistant S. aureus identification system with 63 of 69 (91%) stock isolates of S. aureus yielding a positive result in 4 h, 66 of 69 (95%) yielding a positive result in 5 h, and 68 of 69 (99%) yielding a positive result in 6 h. The corresponding percentage agreements at 4, 5, and 6 h for mec gene-positive stock isolates of S. epidermidis were 87, 91, and 96%, respectively.     

Early detection and successful therapy of fulminant chlamydia pneumoniae myocarditis

We report a case involving a young female patient with Chlamydia pneumoniae myocarditis who required assist device therapy for acute heart failure. Early diagnosis was provided by endomyocardial biopsy, and tailored antibiotic therapy facilitated quick recovery of myocardial function. This is the first case to report detecting C pneumoniae as the pathogen responsible for fulminant myocarditis while the patient was still alive and to report long-term follow-up data.     

Significant hybridization differences in comparative genomic hybridization due to nucleotides used for DNA labelling and to DNA chosen for cohybridization.

OBJECTIVE: Comparative genomic hybridization (CGH) has been established as an informative technique in genetic analysis. However, differences in the ratio of hybridization intensities were reported for particular chromosomes, which may affect CGH results. The aim of this study was to define these differences in more detail. For this purpose, CGH results of 70 samples of bone marrow cells (BMC) with normal karyotype in conventional cytogenetics (CC) were evaluated using seven different reference DNAs and two different DNA labeling systems. METHODS AND RESULTS: CGH using fluorochrome-conjugated nucleotides for DNA labeling indicated signal deviations in 21/70 BMC samples. Deviations affected chromosomes 1 (n = 21), 2 (n = 11), 4 (n = 11), 5 (n = 9), 6 (n = 7), 7 (n = 2), 8 (n = 2), 12 (n = 5), 13 (n = 15), 14 (n = 1), 16 (n = 17), 17 (n = 11), 19 (n = 21), 20 (n = 12), and/or 22 (n = 17). None of the imbalances were confirmed by fluorescence in situ hybridization (FISH). Using digoxigenin and biotin-conjugated nucleotides in exemplary cases (n = 5) led to the disappearance of the signal deviations. Repeated CGH experiments using seven different reference DNAs showed remarkable variations in the signal deviations. CONCLUSION: Hybridization differences depend not only on the hapten or fluorochrome-labeled nucleotides used for DNA labeling, but also on the reference DNA chosen. Therefore, close control of CGH experiments is mandatory, and additional techniques such as FISH should be performed to confirm the results obtained by CGH.     

Apparent superiority of H2-receptor stimulation and simultaneousβ-blockade over conventional treatment withβ-sympathomimetic drugs in post-acute myocardial infarction: Cardiac effects of impromidine — a new specific H2-receptor agonist — in the surviving catecholamine-insensitive myocardium

Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to -adrenergic and H₂-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [³H]-dihydroalprenolol ([³H]-DHA), [³H]-methyl-tiotidine ([³H]-TIOT) and [³H]-quinuclidinyl benzilate ([³H]-QNB) to cardiac ₁-, H₂- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of-sympathomimetic, in contrast to administration of prenalterol and the conventional therapy with -sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventriculardP/dt _max was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (K _D) of the remaining -receptors while specific [³H]-TIOT- and [³H]-QNB-binding was unchanged.Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [³H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal -receptors. Furthermore, treatment with H₂-agonists in combination with -blocking agents may have beneficial effects, whereas conventional therapy with -sympathomimetic drugs tends to worsen the already depressed function of the -adrenergic stimulation mechanism.Supported by grants Ba 666/1 and Ba 666/2-2 from the Deutsche Forschungsgemeinschaft (DFG).Data presented in this paper are part of a doctoral thesis by Dr S.B. Felix.     

Persistent centrilobular necroses in hepatic allografts

A biopsy study of 60 allografts from 53 patients after orthotopic liver transplantation (OLT) revealed prominent centrilobular necrosis (CN) in 18% of the grafts that were suitable for analysis. The lesions often had a "punched-out" appearance, sometimes with unusual features such as giant cell formation. Persistent CN developed 4 weeks to 6 months after OLT, and persisted in two cases for 2 years and longer. In some instances, CN disappeared or healed by scarring. We found no association between CN and rejection arteritis or arteriopathy. Ductopenic (chronic) rejection subsequently occurred in six of eight livers with CN. Overall, patients with persistent CN had a worse prognosis than control patients. A comparison of cases with matched controls failed to reveal significant differences with respect to perioperative factors such as ischemia time, immunologic test results such as lymphocyte crossmatches, drug administration--in particular, of azathioprine, frequency of cellular (acute) rejection or infection episodes, or frequency of complications affecting major hepatic vessels or bile ducts. Morphologic evidence suggests that in some instances, rejection-induced endotheliitis/phlebitis of hepatic vein branches may lead to sinusoidal outflow blockage, sinusoidal dilatation, and dropout of hepatic cell plates. Although potentially reversible conditions such as ischemia or adverse drug reactions are among the possible causes of CN, severe rejection leading to ductopenia appears to be the most important underlying condition. Thus, presence of CN in repeated biopsy specimens from allografts should be considered a warning sign of irreversible rejection.     

Differences in airway remodeling between subjects with severe and moderate asthma

BACKGROUND: Airway remodeling in asthma comprises a range of structural changes. Several studies have suggested an association between these changes and disease severity. The relationship between the extent of remodeling and lung function is not well defined. OBJECTIVE: We sought to contrast the structural changes in the airways of well-defined groups of subjects with severe and moderate asthma and to correlate the extent of remodeling with disease severity. METHODS: Endobronchial biopsy specimens were obtained from 15 subjects with severe and 13 subjects with moderate asthma. Epithelial integrity, cell-layer areas, subepithelial fibrosis, and the distance between epithelial and airway smooth muscle (ASM) layers were measured by means of image analysis. Collagen was identified by using Van Giesen stain, and ASM was defined by using smooth muscle alpha-actin immunostaining. Specific immunostains were performed for the evaluation of RANTES, IL-8, and eotaxin expression as markers of ASM phenotype. RESULTS: ASM area was greater in subjects with severe (0.24+/- 0.03 mm(2)) than in subjects with moderate (0.05+/- 0.01 mm(2)) asthma (P<.001). The distance between the epithelial and ASM layers was less in the severe group (0.12+/- 0.01 mm) than in the moderate group (0.24+/- 0.02, P<.001). A trend toward greater subepithelial fibrosis in subjects with severe asthma did not reach statistical significance. IL-8 and eotaxin expression, but not RANTES expression, were increased in the ASM of subjects with severe asthma compared with in subjects with moderate asthma. CONCLUSION: Smooth muscle alteration is the key structural change that distinguishes severe from moderate asthma, and phenotypic change in ASM might contribute to the difficulty in obtaining adequate control in some subjects with severe asthma.     

Antithrombotic properties of trillium coated connectors

Trillium coating (Medtronic Inc., Minneapolis, MN) offers, in addition to the presence of heparin, endothelium-like properties of its negatively charged surface. Its thromboresistant properties on coated connectors are tested here and compared with uncoated standard connectors, as well as with the Carmeda BioActive surface (CBAS) heparin surface coating. A partial cardiopulmonary bypass bovine model (body weight 68 +/- 5 kg) was selected, and the surfaces were exposed to the blood stream (pump flow 3.5 L/min) for up to 350 minutes without systemic heparinization. Thereafter, another set of samples was exposed to stagnant blood for 20 minutes. Besides hemodynamic, hematologic, and biochemical analyses, the macroscopic appearance of 45 blood exposed surface samples were graded semiquantitatively on a scale of 0 to 10: no macroscopic deposits = grade 0, one spot (1 mm diameter) = grade 1, two spots = grade 2, five or more spots = grade 5, 10% of the surface covered with clots = grade 6, 100% covered = grade 10. When exposed to blood flow, Trillium and CBAS coatings showed a statistically significant (p = 0.03) better thromboresistance (score: 0 +/- 0 for both) than uncoated connectors (score: 0.8 +/- 1.5) in this nonheparinized model. The same holds true when the connectors were exposed to stagnant blood (score: 0 +/- 0 for both coatings vs 4.3 +/- 2.8 for controls; p = 0.03). Therefore, Trillium coating exhibits significant antithrombotic properties that outperform standards for connectors used in clinical perfusion.     

Fluorescence in situ hybridization reveals closely correlated results in cytological and histological specimens of hematological neoplasias compared to conventional cytogenetics.

OBJECTIVES: Fluorescence in situ hybridization (FISH) has become a useful tool to identify chromosomal aberrations in non-dividing cells. Numerous studies have compared chromosomal banding analysis (CBA) and FISH on fixed cultured bone marrow cells. However, up to now, there has been no study comparing two main sources of diagnostic material, i.e. bone marrow aspirates and trephine biopsies. We therefore analyzed these materials by FISH in comparison with CBA. METHODS: CBA revealed chromosomal aberrations in 18 patients suffering from myelodysplastic syndrome (n = 13), acute myeloid leukemia (n = 3), or chronic myeloproliferative syndrome (n = 2). FISH was performed on fixed cultured bone marrow cells, aspirates and trephine biopsies from each patient. RESULTS: Percentages of aberrant cells in the different materials correlated highly with Pearson values of 0.909 for biopsy/fixed cultured cells (p < 0.001), 0.830 for biopsy/aspirate (p < 0.001) and 0.768 for aspirate/fixed cultured cells (p < 0.001). Moreover, in bone marrow biopsies peritrabecular and central intertrabecular areas yielded very similar FISH results with a high correlation (r = 0.968, p < 0.001). FISH revealed a lower proportion of aberrant cells than CBA in 90% of the specimens. CONCLUSIONS: In summary, the different materials available for the FISH examination are comparable in sensitivity and show similar quantitative results. Therefore, the use of biopsy sections for the routine FISH examination of chromosomal abnormalities is a valid method.     

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.