PD0084430: a non-nucleoside inhibitor of human cytomegalovirus replication in vitro.

Human cytomegalovirus (HCMV) is a major opportunistic pathogen in immunocompromised individuals. Current therapies target viral DNA replication and accumulate mutations that yield cross-resistance among the approved drugs. A novel, non-nucleoside inhibitor of HCMV replication, PD0084430, was identified in a screening assay using the HCMV beta-galactosidase recombinant RC256. The EC(50) for PD0084430 by inhibition of beta-galactosidase production is 1+/-0.7 microM. This antiviral activity was confirmed by yield reduction and plaque reduction assays using HCMV strain AD169. The TC(50) of PD0084430 as measured by (4C)thymidine incorporation is approximately 30 microM and by XTT is approximately 90 microM. The TC(50) for inhibition of cellular proliferation is approximately 20 microM. Time of addition experiments displayed a similar drop in efficacy for both PD0084430 and GCV when added after the onset of viral DNA replication. The transcomplementation assay for viral DNA replication, using a transfected ori(Lyt) containing plasmid, confirmed that viral DNA synthesis was inhibited at the same concentrations that showed antiviral activity. Western blots showed no apparent block of immediate early or early gene expression. Two ganciclovir (GCV) resistant isolates of HCMV tested showed no cross-resistance to PD0084430. These data suggested a potentially promising novel compound that inhibited HCMV at or before viral DNA replication. However, in vivo testing in mice dosed either orally or intraperitoneally showed rapid glucuronidation on the -OH group. SAR studies on this backbone showed that the -OH group was essential for the antiviral activity in vitro.

Disseminated haemangiosarcoma in an eastern barred bandicoot (Perameles gunnii).

A captive adult male Eastern barred bandicoot (Perameles gunnii) presented with three palpable subcutaneous masses in November 1998. A diagnosis of haemangiosarcoma was made based on histological examination of one excised mass. Euthanasia of the animal was performed 11 days postsurgery and a proliferative lesion in the paralumbar musculature and similar, smaller proliferative lesions surrounding the right popliteal lymph node and in the ventricular wall of the heart were found. Metastatic lesions were found in the liver and lung. The histological features of the neoplastic tissues supported the diagnosis of a poorly differentiated, disseminated haemangiosarcoma. This is the first reported case of haemangiosarcoma in the Eastern barred bandicoot.

Plasma amino-terminal pro-brain natriuretic peptide: a novel approach to the diagnosis of cardiac dysfunction.

BACKGROUND: Heart failure is a common cause of hospitalization and death across the industrialized world. Improving the diagnosis and care of patients with heart failure is therefore likely to have a major impact on morbidity, mortality, and health care costs. METHODS AND RESULTS: To determine the relation between cardiac function and plasma levels of amino-terminal brain natriuretic peptide precursor (NT-proBNP), plasma NT-proBNP levels and ventricular function (by radionucleotide ventriculography) were measured in healthy patients, patients with renal failure, patients with recent myocardial infarction, and patients investigated for cardiorespiratory symptoms. Plasma NT-proBNP levels were greater in healthy women (median, 1.5 fmol/mL; range, 1.0 to 13.8 fmol/mL; n = 34) than healthy men (median, 1.0 fmol/mL; range, 1.0 to 3.3 fmol/mL; n = 33; P = .012). NT-proBNP levels were elevated in subjects with renal failure (geometric mean, 314 fmol/mL; range, 18 to 5,800 fmol/mL) and were related to left ventricular ejection fraction (LVEF) (r = -0.86; P < .0001; n = 19). NT-proBNP levels were also related to LVEF in patients with recent myocardial infarction (r = -0.62; P = .0003; n = 29) and those investigated for cardiorespiratory symptoms (r = -0.56; P < .0001; n = 129). Applying an upper limit of normal of 5 fmol/mL for men and 15 fmol/mL for women (specificity, 100%), elevated plasma NT-proBNP levels had 100% sensitivity for the detection of LVEF less than 45% after myocardial infarction and 97% sensitivity for the detection of LVEF less than 45% in patients investigated for cardiorespiratory symptoms. NT-proBNP levels were also elevated in 87% of the patients with normal systolic function (LVEF > or = 45%) after myocardial infarction and in 87% of the patients investigated for cardiorespiratory symptoms with heart failure and normal systolic function (LVEF > or = 45%). CONCLUSIONS: Plasma NT-proBNP level is a sensitive indicator of cardiac dysfunction, both in the presence and absence of systolic dysfunction, and may prove to be a useful tool for the identification and management of cardiac dysfunction in the general community.

Psychological influences on the perception of immune function.

BACKGROUND: Perception of deficiencies in immunity are common in a number of patient complaints. However, little is known about the way in which individuals form perceptions about the competence of their immune system. In two studies we examined the relationship between subjects' perceptions of their immune functioning, physical symptoms, mood and measures of immunity. METHODS: In Study 1, 20 healthy volunteers completed global ratings of their immune system functioning, as well as mood and symptom reports, twice a week for 5 weeks. At the same time, blood samples were taken to assess serum IgA, IgG, and IgM antibodies. In Study 2, another sample of 58 subjects completed the same measures weekly for 5 weeks and their blood was tested for concentrations of CD3, CD4, CD8, and CD16 lymphocytes. RESULTS: We found perceptions of immune functioning to be unrelated to the concentrations of serum antibodies or blood lymphocytes. Immune perceptions were strongly related to mood and in particular, feelings of fatigue and vigour. The experience of recent physical symptoms, while not as strong as mood variables, was also important in perceptions of immune functioning. CONCLUSIONS: Mood seems to be an important determinant in the perception of immune function, and complaints about immune dysfunction in clinical situation should be investigated with this possibility in mind.

Positive effects of illness reported by myocardial infarction and breast cancer patients.

This study investigated the positive effects or changes that patients identified in their lives following a myocardial infarction (MI) or breast cancer. One hundred and forty-three patients were assessed in hospital following a first-time MI and 52 breast cancer patients were assessed on referral for radiotherapy. Approximately 3 months later both groups were asked if any positive changes had taken place in their lives following their illness. Patients' written responses to a single open-ended question were read independently by three judges who identified seven major positive themes. Approximately 60% of each patient group reported positive changes from their illness and the reporting of such changes was unrelated to illness severity. The most common theme reported by MI patients was healthy lifestyle change (68%), whereas, for breast cancer patients, it was improved close relationships with others (33%). These results are discussed in terms of the way patients make sense of and adapt to chronic illness.

The immunological effects of thought suppression.

Individuals often suppress emotional thoughts, particularly thoughts that arouse negative emotions, as a way of regulating mood and reducing distress. However, recent work has highlighted the complexities and unexpected cognitive and physiological effects of thought suppression. In a study designed to examine the short-term immunological effects of thought suppression, participants wrote about either emotional or nonemotional topics with or without thought suppression. Blood was drawn before and after each experimental session on 3 consecutive days. Results showed a significant increase in circulating total lymphocytes and CD4 (helper) T lymphocyte levels in the emotional writing groups. Thought suppression resulted in a significant decrease in CD3 T lymphocyte levels. The implications of the results for the role of the expression and suppression of emotion in health are discussed.

Reduction in skin reactions to histamine after a hypnotic procedure.

This study sought to test whether a cognitive-hypnotic intervention could be used to decrease skin reactivity to histamine and whether hypnotizability, physiological variables, attitudes, and mood would influence the size of the skin weals. Thirty eight subjects undertook three individual laboratory sessions; a pretest session to determine sensitivity to histamine, a control session, and an intervention session during which the subject experienced a cognitive-hypnotic procedure involving imagination and visualization. Compared with the control session, most subjects (32 of 38) decreased the size of their weals measured during the intervention session, and the differences between the weal sizes produced in the two sessions were highly significant (N = 38; t = 4.90; p < .0001). Mood and physiological variables but not hypnotizability scores proved to be effective in explaining the skin test variance and in predicting weal size change. Feelings of irritability and tension and higher blood pressure readings were associated with less change in weal size (i.e., a continuation of reactivity similar to that found in the control session without the cognitive-hypnotic intervention), and peacefulness and a lower blood pressure were associated with less skin reactivity during the intervention. This study has shown highly significant results in reducing skin sensitivity to histamine using a cognitive-hypnotic technique, which indicates some promise for extending this work into the clinical area.

Disclosure of trauma and immune response to a hepatitis B vaccination program.

This study investigated whether emotional expression of traumatic experiences influenced the immune response to a hepatitis B vaccination program. Forty medical students who tested negative for hepatitis B antibodies were randomly assigned to write about personal traumatic events or control topics during 4 consecutive daily sessions. The day after completion of the writing, participants were given their first hepatitis B vaccination, with booster injections at 1 and 4 months after the writing. Blood was collected before each vaccination and at a 6-month follow-up. Compared with the control group, participants in the emotional expression group showed significantly higher antibody levels against hepatitis B at the 4 and 6-month follow-up periods. Other immune changes evident immediately after writing were significantly lower numbers of circulating T helper lymphocytes and basophils in the treatment group. The finding that a writing intervention influences immune response provides further support for a link between emotional disclosure and health.

Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification.

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.

Conditioning allergic skin responses in humans: a controlled trial.

Classical conditioning of immune responsiveness has been extensively investigated in animals, but few successful studies have been reported with humans. We report the results of a study in which an attempt was made to demonstrate conditioned alteration to an allergic skin test response in humans. For 8 of 10 trials, allergic skin tests were administered to one forearm of volunteer subjects, and saline was administered to the other forearm. For the other two trials, the test substances were covertly switched between arms, but no changes in response resulted from this maneuver that could be ascribed to conditioning.

Effect of vitamin E on adhesion formation.

This study investigated the effects of vitamin E on adhesion formation and fibrosis. Forty-eight adult, virgin, female Sprague-Dawley rats were divided equally into two groups, one of which received 300 mIU/kg vitamin E orally in their daily diet in comparison with the control group, which received the vehicle only. Laparotomy was performed and the uterine horns identified. Denuding of the serosa was performed over a segment of the right horn; bipolar coagulation over a similar area on the left horn was performed. Following the procedure the animals continued on the same diet for three weeks. They were then killed, the adhesions were graded, and microscopic evaluation for fibrosis was accomplished. Although there was a trend of less fibrosis with vitamin E, there was no statistically significant difference, nor was there a difference with respect to a reduction in adhesion formation.

Immediate-type hypersensitivity reactions and hypnosis: problems in methodology.

Hypnosis has been used to ameliorate skin test reactivity in studies dating back to the 1930s. This study using modern methodology and statistical analyses sets out to test the hypothesis that it was possible to decrease reactions to histamine by hypnotic suggestion. Five subjects, all asthmatic and untrained in hypnosis, were given three hypnotic sessions where they were asked to control their reactions to histamine administered by the Pepys technique to forearm skin. These sessions were to be compared with three non-hypnotic sessions. The flare sizes but not wheal sizes were found to be significantly reduced after the hypnosis sessions, compared to sessions without hypnosis. Skin temperature was correlated with the size of reactions. The day upon which the sessions took place contributed significant amounts of the remaining unexplained variance, giving rise to questions about what could cause these day to day changes.

The variability of type I hypersensitivity reactions: the importance of mood.

Immediate (Type I) hypersensitivity skin reactions to allergens or antigens have been used as immune measures that may be subject to intentional modulation. In preliminary experiments using hypnosis we encountered unacceptably large, uncontrollable variability. A method was subsequently devised in which serial, five-fold dilutions of allergen or histamine were administered to the subject's forearm and reactions were recorded photographically on slide film. Areas were determined by computer-assisted image analysis. Seven healthy volunteers were tested for eight sessions (testing included mood scales, blood pressure, pulse and skin temperature). Mean wheal size and titration gradient data from allergen reactions correlated strongly with the psychological factor of liveliness but not stress, although no manipulation of mood was involved. A stepwise regression analysis accounted for 61% of the variance of the allergen mean wheal data, and 31% was from the liveliness factor alone. Thus, the more lively the subject felt, the smaller was the allergic response.

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria.

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.

Effects of aldose reductase inhibition with epalrestat on diabetes-induced changes in rat isolated atria.

1. Isoprenaline and cardiac responsiveness of isolated atria from 2 and 6 week streptozotocin-diabetic rats, and their age-matched controls, was examined. The effects of chronic administration of epalrestat (40 mg/kg orally, by gavage) on diabetes-induced changes were also investigated. 2. Spontaneously beating atria, bathed in either normal or high glucose (30 mmol/L) Krebs' solution, from both 2 and 6 week diabetic rats beat more slowly and with greater force than atria from control rats. These changes in basal parameters were normalized by 2 weeks of insulin (5 U/day s.c.) treatment but not by 2 or 6 weeks of chronic treatment with epalrestat. 3. Isoprenaline (0.1 nmol-0.1 mumol/L) produced concentration-dependent increases in inotropy and chronotropy in atria from both control and diabetic rats. 4. Atria from 2 week diabetic rats displayed decreased sensitivity to the positive inotropic effects of isoprenaline. This change was normalized by chronic insulin treatment but not by chronic epalrestat treatment. 5. Atria from 6 week diabetic rats displayed increased sensitivity to the positive chronotropic effects of isoprenaline which was normalized by epalrestat. 6. These results suggest that changes observed in atria from 2 week diabetic rats may be due to hyperglycaemia per se whereas in atria from 6 week diabetic rats abnormal activity of the polyol pathway may be a contributing factor.

Early maturation of force production in pig tracheal smooth muscle during fetal development.

The contractility of airway smooth muscle is fully established at late term at birth but its responsiveness during fetal life has not been defined. In this study, the contractile force of airway smooth muscle to acetylcholine (ACh), K+ depolarizing solution, and electrical field stimulation (EFS) was measured in tracheas from small fetal pigs. Contraction to either agonist and to EFS was detectable in fetuses of as low as 9 g body weight, which corresponds to approximately 36 days of gestation. Isometric force increased progressively with age, reaching 4.1 +/- 0.4 mN for K+ and 5.8 +/- 0.5 mN for ACh (10(-4) M) at 600 g fetal weight (90 days). However, when normalized for cross sectional area of smooth muscle, the stress was essentially the same from 17- to 600-g fetuses. (K+: 17 g = 74.4 +/- 10.6 mN/mm2, 600 g = 89.3 +/- 13.0 mN/mm2; ACh [10(-4) M]: 17 g = 76.3 +/- 16.0 mN/mm2, 600 g = 127.0 +/- 13.0 mN/mm2). The sensitivities of the various groups to ACh were not significantly different (e.g., EC50: 30 g = 4.0 +/- 0.2 x 10(-6) M, 600 g = 3.7 +/- 1.1 x 10(-6) M). EFS produced frequency-dependent contractile responses in all groups. With increasing fetal size, there was a corresponding increase in force. When this force was normalized to a maximum ACh response (10(-4) M), there was no significant difference between groups of fetuses. Histologic examination showed the major tissue components of the trachea were present in fetuses above 7 g. Immunocytochemistry detected myosin, caldesmon, and filamin in the smooth muscle from fetuses of 7 g and above, showing that contractile and actin-binding proteins were present from a very early age. It is concluded that smooth muscle contractile function is well developed very early in fetal life in pigs.

The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae.

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.