Endocarditis caused by culture-negative organisms visible by Brown and Brenn staining: utility of PCR and DNA sequencing for diagnosis.

Two cases of culture-negative endocarditis with cocci seen in valve vegetations are presented. The organisms were identified by molecular analysis using broad-range PCR primers complementary to the 16S rRNA gene, sequencing, and database search using BLAST software. The results and utility of this method are discussed.

Combined treatment with 2'-nor-cGMP and ganciclovir against cytomegalovirus infection in a guinea pig model.

The combination 2'-nor-cGMP/DHPG at fixed ratios 1:5, 1:10 and 1:20 showed synergistic antiviral effects against GPCMV replication in vitro with CI value < 1. In vivo, a fixed ratio of 1:10 at three different dosage levels of 1.25/12.5 mg, 2.5/25 mg and 5/50 mg/kg/day 2'-nor-cGMP/DHPG combination showed only additive results when compared with each drug alone. However, synergistic antiviral effects were obtained when infected guinea pigs were treated with 2'-nor-cGMP/DHPG combination 2.5/10 mg/kg/day (1:4). A significantly lower GPCMV infectivity titer was noted in the salivary gland, lung and spleen of infected guinea pigs treated with the combination of 2'-nor-cGMP/DHPG 2.5/10 mg/kg/day, as compared to animals treated with a corresponding dose of each drug alone. In addition, GPCMV-infected animals treated with the latter combination showed increased body weight than when either drug was used alone. Histopathologically, each drug alone reduced the viral induced changes in the lung and spleen, but the combination therapy reduced these changes still further. Toxic changes seen in the kidney and bone marrow of infected animals treated with 2'-nor-cGMP, 2.5 mg/kg/day were not significantly increased when DHPG 10 mg/kg/day was added to the regimen. Therefore, combined treatment with 2'-nor-cGMP/DHPG in appropriate concentration is more helpful for acute cytomegalovirus infection in guinea pigs than when either drug was used alone.

Demonstration of cytomegalovirus and retrovirus pseudotypes in cultured guinea pig cells.

A mixed viral infection with a cytomegalovirus and a retrovirus in cultured guinea pig embryo (GPE) cells was investigated. The expression of an endogenous guinea pig retrovirus (GPRV) in cultured guinea pig cells was induced by a medium containing 5-bromo-2'-deoxyuridine and dexamethasone. When the induced GPE cells were superinfected with a guinea pig cytomegalovirus (GPCMV), pseudotype virions were observed. Morphological characterization of both viruses and their locations within infected cells was achieved by examination of thin sections of infected cells with transmission electron microscopy. Immunolabeling with colloidal gold particles, 5 or 15 nm in size, permitted the identification of each virus type using GPCMV- or GPRV-specific polyclonal antibodies and the detection of a population of GPCMV and GPRV particles which expressed antigens of both viruses on their envelopes. Enhanced reverse transcriptase activity of GPRV and reduced infectivity titers of GPCMV was noted in dually infected cultures. These data suggest that interaction between GPCMV and GPRV had occurred in dually infected GPE cells and that expression of GPRV was enhanced.

Combined antiviral effects of paired nucleosides against guinea pig cytomegalovirus replication in vitro.

Several promising antiviral nucleosides have been tested in paired combinations against guinea pig cytomegalovirus (GPCMV) replication in guinea pig embryo (GPE) cells by plaque reduction assay; these are [9-(2-hydroxy-1-3-2-dioxaphosphorinan-5-yl)oxymethyl]-guanin e P-oxide (2'nor-cGMP, compound 164), [4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo(2,3-d)pyrimidine] (compound 102), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV) and 3'-azido-3'-deoxythymidine (zidovudine, AZT). Various degrees of interactions were observed; i.e. synergistic reactions were noted in the presence of compound 164/compound 102 and compound 164/DHPG combinations at all concentrations tested. HPMPC/DHPG combinations were synergistic at relatively lower concentrations of DHPG, but became antagonistic as the concentration of DHPG increased. Combinations of compound 164/ACV and DHPG/AZT were antagonistic.

delta-Aminolevulinic acid dehydratase in rat liver: studies on the effects of ethanol, acetaldehyde, and B6 vitamers.

Because ethanol ingestion lowers delta-aminolevulinic acid dehydratase (ALAD) activity in liver and red cells, effects of ethanol and acetaldehyde on ALAD in rat liver cytosol were studied. When added to the assay mix, as little as 0.5 mmol/L acetaldehyde competitively inhibited ALAD even in the presence of dithiothreitol, a sulfhydryl reagent. ALAD activity also fell when undiluted cytosol was incubated at 37 degrees with as little as 0.25 mmol/L acetaldehyde for 8 hours before enzyme assay. Inactivation of ALAD by acetaldehyde was prevented by the metabolic inhibitor NaF but not by the aldehyde dehydrogenase inhibitor cyanamide. Incubation of undiluted cytosol with 20 mmol/L ethanol also decreased ALAD activity, but addition of ethanol to the assay mix had no effect. Ethanol-mediated inactivation of ALAD was reduced by inhibition of alcohol dehydrogenase with 4-methylpyrazole, but ALAD activity was not decreased by incubation of undiluted cytosol with acetate or sorbitol or by addition of acetate to the assay mix. The aldehydic B6 vitamers, pyridoxal and pyridoxal phosphate, also inhibited ALAD activity when added to the assay mix. However, these vitamers increased ALAD activity and decreased acetaldehyde-mediated inactivation of ALAD when incubated for 8 hours with undiluted cytosol. We conclude that (1) acetaldehyde decreases ALAD activity both by competitive inhibition with substrate and by inactivation of enzyme protein and that (2) inactivation of ALAD by acetaldehyde may require nonoxidative metabolism of acetaldehyde. The net pharmacologic effect of B6 vitamers on ALAD activity and on inactivation of ALAD by acetaldehyde remains to be determined.

Studies on the mechanism of acetaldehyde-mediated inhibition of aspartate aminotransferase (GOT) in human erythrocytes.

The mechanism of acetaldehyde-mediated GOT inhibition was studied in human red cells. In the GOT assay mix, acetaldehyde competitively inhibits activation of apoGOT by pyridoxal phosphate and pyridoxamine phosphate. However, Ki values are 100-1000 times greater than Km values for these B6 vitamers. Moreover, incubation of undiluted lysates with acetaldehyde at 37 degrees inhibits GOT activity without increasing apoGOT levels and without altering affinity of apoGOT for either B6 coenzyme. In undiluted lysates, inhibition is not prevented by disulfiram. However, incubation at 4 degrees prevents both acetaldehyde metabolism and GOT inhibition while preincubation with NaF prevents GOT inhibition without affecting acetaldehyde disappearance. The effect of NaF is completely reversed by pyruvate but only partially reversed by NADH. Glyceraldehyde 3-phosphate, the only glycolytic intermediate which directly inhibits GOT, does not reverse the NaF effect. Thus, inhibition of GOT by acetaldehyde (a) requires nonoxidative metabolism of acetaldehyde and (b) is not mediated either by glycolytic substrates or by impaired binding of B6 vitamers to the GOT apoenzyme. Since NaF had no effect on a lysate deficient in glucose 6-phosphate dehydrogenase, the hexose monophosphate shunt may play a role in acetaldehyde-mediated GOT inhibition.

Inhibition of rat liver transaminases by low levels of acetaldehyde and the pharmacologic effects of B6 vitamers.

To better define the significance and mechanism of acetaldehyde-mediated transaminase inhibition, acetaldehyde metabolism was studied in rat liver homogenates and cytosols. When either preparation was incubated at 37 degrees with 1.5 mM acetaldehyde for 4 hr, acetaldehyde levels fell rapidly in the first 30 min and little inhibition of aspartate aminotransferase (GOT) or alanine aminotransferase (GPT) resulted. In contrast, incubation with 50 mM ethanol also resulted in a peak acetaldehyde level of 1.0 to 1.5 mM by 2 hr, but this level was then maintained for the next 2 hr and transaminases were inhibited by 20-35%. Sequential addition of low dose (125-250 microM) pulses of acetaldehyde to rat liver preparations resulted in a progressive decrease in the rate of acetaldehyde disappearance. When the pulsing schedule was adjusted accordingly to maintain acetaldehyde levels between 50 and 250 microM for 8 hr, transaminases were again inhibited by 20-40%. Finally, addition of 1-5 mM pyridoxal and pyridoxal 5'-phosphate, aldehydic B6 vitamers, to cytosols 2-4 hr after pulsing with acetaldehyde was begun, almost completely prevented further transaminase inhibition. In contrast, the non-aldehydic B6 vitamers, pyridoxine, pyridoxamine and pyridoxamine 5'-phosphate, did not affect acetaldehyde-mediated transaminase inhibition. These findings suggest that (1) prolonged exposure to low levels of acetaldehyde impairs acetaldehyde metabolism in rat liver homogenates and cytosols; (2) acetaldehyde toxicity may be more dependent on sustained exposure to acetaldehyde than on the peak level of acetaldehyde attained; and (3) aldehydic B6 vitamers can modify on-going acetaldehyde-mediated transaminase inhibition.

Assessment of leukocyte alkaline phosphatase by image analysis.

We have shown that it is possible to automate the assessment of leukocyte alkaline phosphatase by using an azo dye cytochemical staining procedure and a commercial, highly sophisticated image analysis instrument originally designed specifically as a differential white cell counter. The data to date indicate that values obtained by this approach are at least as precise and accurate as current manual techniques. Instrumental analysis avoids the subjectivity associated with manual interpretation of staining intensity and should permit meaningful interlaboratory comparisons. The stability of the stained smears upon exposure to immersion oil or Polymount mounting medium proved to be an unexpected bonus. In addition to such functional data on leukocytes as illustrated by this report, these instruments, with appropriate staining methods and software, can also provide clinically useful quantitative data on red cells, as have been described for reticulocytes. We hope to see more clinical applications in the future for these expensive and target-oriented image analysis instruments. They are capable of automatically providing objective quantitative information on a cell by cell basis--providing feature data that cannot be obtained by other means.

Relationship of reticulocyte age to polychromasia, shift cells, and shift reticulocytes.

Polychromatophilic erythrocytes on Wright-stained blood smears represent young reticulocytes. Ratios of polychromatophilic cells to total reticulocyte counts have been used to estimate marrow response to erythropoietin stimulation. These ratios, however, require both accurate counts of polychromatophilic cells on Wright-stained blood smears and reticulocytes on supravitally stained blood smears. Data from this study indicated that reticulocytes of Heilmeyer groups I, II, and III best represent polychromatophilic cells. Group III reticulocytes, however, were found in normal circulation and were difficult to distinguish from group IV reticulocytes. Groups I and II were not found in normal circulation and were easily identified on routine reticulocyte preparations. The term "shift reticulocyte" is proposed for reticulocytes of groups I and II only. The present study suggests that the "shift reticulocyte count," expressed as percent of 100 reticulocytes, is a more useful indicator of marrow response to anemia than total reticulocyte counts.