The value of uncertainty in critical illness? An ethnographic study of patterns and conflicts in care and decision-making trajectories.

BACKGROUND: With increasingly intensive treatments and population ageing, more people face complex treatment and care decisions. We explored patterns of the decision-making processes during critical care, and sources of conflict and resolution. METHODS: Ethnographic study in two Intensive Care Units (ICUs) in an inner city hospital comprising: non-participant observation of general care and decisions, followed by case studies where treatment limitation decisions, comfort care and/or end of life discussions were occurring. These involved: semi-structured interviews with consenting families, where possible, patients; direct observations of care; and review of medical records. RESULTS: Initial non-participant observation included daytime, evenings, nights and weekends. The cases were 16 patients with varied diagnoses, aged 19-87 years; 19 family members were interviewed, aged 30-73 years. Cases were observed for <1 to 156 days (median 22), depending on length of ICU admission. Decisions were made serially over the whole trajectory, usually several days or weeks. We identified four trajectories with distinct patterns: curative care from admission; oscillating curative and comfort care; shift to comfort care; comfort care from admission. Some families considered decision-making a negative concept and preferred uncertainty. Conflict occurred most commonly in the trajectories with oscillating curative and comfort care. Conflict also occurred inside clinical teams. Families were most often involved in decision-making regarding care outcomes and seemed to find it easier when patients switched definitively from curative to comfort care. We found eight categories of decision-making; three related to the care outcomes (aim, place, response to needs) and five to the care processes (resuscitation, decision support, medications/fluids, monitoring/interventions, other specialty involvement). CONCLUSIONS: Decision-making in critical illness involves a web of discussions regarding the potential outcomes and processes of care, across the whole disease trajectory. When measures oscillate between curative and comfort there is greatest conflict. This suggests a need to support early communication, especially around values and preferred care outcomes, from which other decisions follow, including DNAR. Offering further support, possibly with expert palliative care, communication, and discussion of 'trial of treatment' may be beneficial at this time, rather than waiting until the 'end of life'.

Absence of adenosine deaminase activity in a mammalian cell line transformed by Rous sarcoma virus.

No detectable adenosine deaminase activity was found in whole cells or 105,000g cytosol preparations of B-mix K-44/6 cells when either [3H]adenosine or [3H]arabinosyladenine was used as substrate. When grown in tissue culture medium supplemented with horse serum these cells provide a deaminase-free system not requiring the use of an adenosine deaminase inhibitor.

Tritiated thymidine incorporation does not measure DNA synthesis in ribavirin-treated human cells.

When the incorporation of tritiated thymidine into acid insoluble material was measured, ribavirin appeared to be a potent inhibitor of DNA synthesis in KB cells and human lymphocytes. Inhibition was nearly 100-fold less, however, when DNA synthesis was measured by incorporation of phosphorus-32-labeled phosphate or by DNA fluorescence. The potent inhibition detected by incorporation of tritiated thymidine into DNA actually was the result of a potent effect on the labeling of deoxythymidine triphosphate, not on the synthesis of DNA.

The views of patients with advanced cancer regarding participation in serial questionnaire studies.

Longitudinal research helps to clarify changing needs and the timing of treatments and referral but is hampered by poor recruitment and retention of participants. We explored, using semi-structured interviews in a cross-sectional design, the views and preferences of patients with advanced cancer on taking part in planned longitudinal questionnaire-based research studies. Patients with advanced lung and colorectal cancer were recruited from outpatient clinics in a London hospital. Semi-structured interviews were undertaken to explore their views about taking part in a specific future questionnaire study and their preferences regarding format. In all, 20 of 47 patients initially identified were recruited. Their preferences for the planned questionnaire study were for face-to-face interviews undertaken at home from late morning onwards with recontact at a mean of 6 weeks. Fluctuating symptom control needs could result in unexpected admission to or discharge from hospital. Developing flexible and responsive recruitment procedures is vital to retain patient participation as more than one contact might be required to successfully conclude an interview.

Deoxyadenosine antagonism of the antiviral activity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosylhypoxanthine.

Deoxyadenosine but not adenosine reversed the antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) and 9-beta-D-arabinofuranosylhypoxanthine (ara-H) when used in the presence of coformycin, an inhibitor of adenosine deaminase. In suspension cultures of KB cells, 10 muM ara-A inhibited the replication of herpes simplex virus type 1 by 80%. Concomitant addition of 50 muM deoxyadenosine reduced the antiviral activity of 10 muM ara-A to only 40% inhibition. Adenosine failed to antagonize the antiviral activity. In monolayer cultures of KB cells, the 50% inhibitory concentration of ara-A was increased from 1.5 to 2.9 muM by 2 muM deoxyadenosine and to 8.5 muM by 10 muM deoxyadenosine. Analysis of the dose-response data by a double reciprocal plot method indicated that the antagonism was competitive. The antiviral activity of ara-H also was antagonized by deoxyadenosine. The 50% inhibitory concentration of ara-H was increased from 42 muM to 70, 91, or 121 muM by the concurrent addition of 5, 10, or 20 muM deoxyadenosine. Competitive antagonism could not be demonstrated. In the absence of the adenosine deaminase inhibitor, neither ara-A nor ara-H was antagonized by deoxyadenosine. Since such inhibitors were not available unitl recently, previous investigators were unable to observe the antagonistic capacity of deoxyadenosine.

The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway.

It has recently been suggested that bisphosphonates may have direct antitumor effects in vivo, in addition to their therapeutic antiresorptive properties. Bisphosphonates can inhibit proliferation and cause apoptosis in human myeloma cells in vitro. In macrophages, bisphosphonate-induced apoptosis was recently found to be a result of inhibition of the mevalonate (MVA) pathway. The aim of this study was to determine whether bisphosphonates also affect human myeloma cells in vitro by inhibiting the MVA pathway. Incadronate and mevastatin (a known inhibitor of the MVA pathway) caused apoptosis in JJN-3 myeloma cells and inhibited cell proliferation. Geranylgeraniol and farnesol prevented incadronate-induced apoptosis and had a partial effect on cell cycle arrest. MVA and geranylgeraniol prevented mevastatin-induced apoptosis and inhibition of proliferation and completely prevented the effect of mevastatin on the cell cycle. These observations demonstrate that incadronate-induced apoptosis in human myeloma cells in vitro is the result of inhibition of the MVA pathway.

The pharmacology of bisphosphonates and new insights into their mechanisms of action.

Bisphosphonates are chemically stable analogs of inorganic pyrophosphate, which are resistant to breakdown by enzymatic hydrolysis. The biological effects of bisphosphonates on calcium metabolism were originally ascribed to their physico-chemical effects on hydroxyapatite crystals. Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds. Remarkable progress has been made in increasing the potency of bisphosphonates as inhibitors of bone resorption, and the most potent compounds in current use are characterized by the presence of a nitrogen atom at critical positions in the side chain which, together with the bisphosphonate moiety itself, seems to be essential for maximal activity. As a class the bisphosphonates offer a very effective means of treating Paget's disease.

7-Aminoquinolines. A novel class of agents active against herpesviruses.

A series of 7-aminoquinoline derivatives was synthesized and evaluated for their capacity to produce cytotoxicity in KB cells and to inhibit the replication of herpes simplex virus (HSV) type 1. All compounds tested inhibited the replication of HSV-1 with 50% inhibitory concentrations in the range of 2-50 micrograms/mL. The antiviral activity of many compounds, however, was separated from cytotoxicity to replicating uninfected cells by only two- to fivefold higher than those required for antiviral activity. Nonetheless, six compounds (10, 28, 29, 32, 34, and 36) were identified in which the separation was greater than fivefold. All compounds examined were more potent inhibitors of viral DNA synthesis than the cellular DNA synthesis.

Phosphorylation of triciribine is necessary for activity against HIV type 1.

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.

Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone.

The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (IC50 values) range from 2 to 13 microM. Four of the five derivatives also were inhibitors of the host cell reductase (IC50 values = 7-34 microM). A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme (IC50 = 340 microM). Four of six compounds were preferential inhibitors of the viral reductase based on a comparison of IC50 values (5- to greater than 85-fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs.

Metabolism of arabinosyladenine in herpes simplex virus-infected and uninfected cells. Correlation with inhibition of DNA synthesis and role in antiviral selectivity.

The metabolism of 9-beta-D-arabinofuranosyladenine (ara-A, vidarabine) and its effects on DNA synthesis were compared in uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells. In the absence of an inhibitor of adenosine deaminase, ara-A was deaminated to 9-beta-D-arabinofuranosylhypoxanthine and phosphorylated to ara-A-5'-mono-, di- and triphosphates in both types of cells. When an inhibitor of adenosine deaminase (coformycin) was added to cell cultures, nucleotides were the only metabolites detected--primarily the 5'-triphosphate of ara-A (aATP). Detailed studies performed in the presence of coformycin established that the net rate and extent of aATP formation were the same in uninfected and HSV-1-infected cells. After a 12-hr exposure to 50 microM ara-A, intracellular concentrations of aATP were approximately 40 microM. Levels of aATP correlated directly with inhibition of total DNA synthesis. Approximately 0.7 microM aATP was required for 50% inhibition of total DNA synthesis in both uninfected and HSV-1-infected cells. Following removal of ara-A-containing culture medium, aATP levels in uninfected cells declined with a half-life of 3.2 hr. In marked contrast, the half-life in HSV-1-infected cells was 9.3 hr; this may explain why as little as a 3-hr exposure to ara-A resulted in a significant HSV-1 titer reduction. Taken together, the data show that when ara-A was removed from culture medium, levels of aATP persisted longer in HSV-1-infected cells thereby prolonging antiviral activity. This effect could be important in vivo where levels of ara-A oscillate with dosing schedule.

Clinical disorders of bone resorption.

Clinical disorders in which bone resorption is increased are very common and include Paget's disease of bone, osteoporosis, and the bone changes secondary to cancer, such as occur in myeloma and metastases from breast cancer. Clinical disorders of reduced bone resorption are less common and often have a genetic basis, e.g. in osteopetrosis, and in pycnodysostosis due to cathepsin K deficiency. Bone is metabolically active throughout life. After skeletal growth is complete, remodelling of both cortical and trabecular bone continues and results in an annual turnover of about 10% of the adult skeleton. The commonest disorder of bone resorption is osteoporosis, which affects one in three women over 50 years. Its pathophysiological basis includes genetic predisposition and subtle alterations in systemic and local hormones, coupled with environmental influences. Treatment depends mainly on drugs that inhibit bone resorption, either directly or indirectly. This includes bisphosphonates, oestrogens, synthetic oestrogen-related compounds (SERMs--selective oestrogen receptor modulators) and calcitonin. The most widely used drugs for all disorders of increased bone resorption, including osteoporosis, are the bisphosphonates. Recent elucidation of their mode of action, together with the rapidly increasing knowledge of regulatory mechanisms in bone biology, offers many opportunities for the development of new therapeutic agents.

A three-dimensional model to analyze drug-drug interactions.

Nearly four generations of investigators have studied combined drug effects. Their methods of generating and analyzing data have changed dramatically over the years but the basic problem has not. This review examines the inherent difficulties in analyzing combined drug effects and evaluates modern methods of describing these interactions. Researchers have traditionally used two-dimensional (2-D) methods to approximate the actual three-dimensional (3-D) nature of drug interactions. We conclude that these 2-D methods are often inadequate when used to analyze synergistic and antagonistic drug interactions in antiviral and anticancer chemotherapy. We propose a direct and pragmatic 3-D approach to the problem, made possible by microcomputers and sophisticated graphics programs. This procedure directly elucidates the shape of the dose-response surface, identifies the regions of statistically significant synergy and antagonism, and quantitates these effects. It also greatly simplifies the problem since a 3-D surface presents complete drug interactions in a way that can be easily interpreted. We will show that understanding the shape of the resulting 3-D surface is essential to an understanding of complex drug interactions. This new method facilitates the rigorous analysis of drug-drug interactions and offers investigators powerful new tools to analyze combinations of antiviral and anticancer drugs.

Inhibitors of thymidylate synthase and dihydrofolate reductase potentiate the antiviral effect of acyclovir.

In cells infected with herpes simplex virus type 1, intracellular dNTP pools increased markedly. Treatment of these cells with 3 microM acyclovir resulted in an additional expansion in pyrimidine deoxyribonucleoside triphosphate pools with dTTP increasing 32-fold and dCTP 8-fold. Both thymidine and deoxycytidine, however, compete with acyclovir for phosphorylation by the viral pyrimidine deoxyribonucleoside kinase and thus reduce the amount of drug that is anabolized to the active form. Theoretically, agents which inhibit thymidylate synthase or dihydrofolate reductase should reduce intracellular pools of thymidine, resulting in the potentiation of the antiviral effects of acyclovir. We explored this strategy by quantitating the synergy produced by combinations of acyclovir and other drugs using three-dimensional dose-response surface methodology (MacSynergy II). Significant synergy was seen with both 5-FdUrd and methotrexate whereas BrVdUrd, 5-CldUrd, 5-IdUrd, and 5-BrdUrd exhibited little to no synergistic activity. It is suggested that inhibitors of thymidylate synthase and dihydrofolate reductase warrant further exploration as potentiators of acyclovir.

Thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model.

A series of 111 thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds were evaluated as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model. All derivatives tested were potent inhibitors of virus replication with mean 50% inhibitory concentrations of 1.1 micrograms/ml for both type 1 and 2 herpes simplex virus. Inhibitory concentrations for cellular protein and DNA synthesis were considerably higher for many compounds resulting in in vitro therapeutic indices ranging from greater than 100 (highly selective) to less than 1 (negatively selective). All compounds were tested for dermal toxicity following topical administration of saturated solutions in 1,3-butanediol to the shaved, depilated skin of guinea pigs. Approximately 50% of the compounds produced slight to no dermal toxicity whereas the remaining compounds produced moderate to severe dermal toxicity. 28 compounds were evaluated in the cutaneous herpes guinea pig model against herpes simplex virus type 1. A number of N4-monosubstituted 2-acetylpyridine thiosemicarbazones produced highly significant reductions in days to healing and lesion score without producing untoward dermal toxicity. Structure-activity relationships revealed that a reduction of the azomethine bond in the molecule (i.e., conversion of a thiosemicarbazone to a thiosemicarbazide) greatly diminished dermal toxicity apparently without producing a proportional decrease in antiviral activity.

The selective inhibition of viral DNA synthesis by chemotherapeutic agents: an indicator of clinical usefulness?

A cell culture system has been utilized to measure the effects of drugs on DNA synthesis in uninfected and HSV-(herpes simplex virus)-infected KB cells. DNA from HSV-infected cells was separated into viral and cellular components by isopycnic centrifugation in CsCl gradients. The amount of [3H]thymidine incorporated into acid-insoluble material was measured in the absence and presence of drugs. Dose-response relationships were established by linearly regressing the probit value of the percent inhibition DNA synthesis against the logarithm of drug concentration. Fifty percent inhibitory (I50) concentrations were interpolated from the corresponding regression lines for inhibition of the following: (i) DNA synthesis is uninfected KB cells, (ii) total DNA synthesis in HSV-infected KB cells (iii) cellular DNA synthesis in HSV-infected cells, and (iv) viral DNA synthesis in HSV-infected cells. We have derived an index (SI, selective index) that quantifies the preferential inhibition of viral or uninfected cellular DNA synthesis. This index can be expressed as SI = log10 I50 concentration for DNA synthesis in uninfected cells divided by I50 concentration for viral DNA synthesis in HSV-infected cells. The SI is positive if viral DNA synthesis is inhibited preferentially and negative if uninfected cellular DNA synthesis is more strongly inhibited. A positive SI value of 0.5 was obtained for the clinically useful antiviral drug arabinosyladenine (ara-A) and a value of 0.4 for its metabolite, arabinosylhypoxanthine (ara-H). Although the adenosine deaminase inhibitor coformycin greatly increased the potency of ara-A, the inhibitor did not increase the selectivity of the drug (SI = 0.3). Stallimycin (distimycin A) (SI = 0.3) and phosphonoacetic acid (SI = 0.3) were similarly effective in preferentially inhibiting the synthesis of HSV DNA. In contrast, arabinosylcytosine (ara-C) and ribavirin inhibited DNA synthesis in uninfected cells to a greater degree than viral DNA synthesis (SI = -0.5 and -1.9, respectively). An analysis of the advantages and limitations of this experimental procedure is made and the suggestion is offered that the in vitro determination of a drug's selective index may be a valid predictor of clinical usefulness.

A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus.

Although the virus yield reduction assay is a powerful technique for evaluating the efficacy of antiviral compounds, it is not routinely utilized due to its labor-intensive nature. This procedure was modified, developed, thereby reducing greatly the time and effort required to perform yield reduction assays. Monolayer cultures of mammalian cells were grown in 96-well microtiter tissue culture plates and infected with virus. Test compounds were added and serially diluted directly with the plates. Following a cycle of virus replication, culture lysates were made and serially diluted in a separate set of uninfected cultures grown in microtiter plates. The cultures were incubated, plaques were enumerated in wells containing 5 to 20 plaques, and virus titers were calculated. To illustrate the use of the assay the known antiviral drugs acyclovir and ganciclovir were evaluated using this procedure. Ninety percent inhibitory concentrations for the respective drugs were 3 microM and 0.7 microM against herpes simplex virus type 1 and 60 microM and 1 microM against human cytomegalovirus.

Anti-tumour activity of bisphosphonates in human myeloma cells.

Multiple myeloma is a haematological malignancy characterized by an expansion of malignant plasma cells within the bone marrow and is frequently associated with bone disease involving the development of osteolytic bone lesions, pathological fractures, osteoporosis and hypercalcaemia. A class of anti-resorptive drugs known as bisphosphonates have been in use to treat osteoclast-mediated bone diseases for the past 3 decades, and are currently proving effective in the treatment of the bone disease associated with multiple myeloma. Recent studies have suggested that bisphosphonate treatment may also result in an improvement in survival in some patients with multiple myeloma. These effects on survival may reflect an indirect effect of the bisphosphonates on tumour growth, via inhibition of osteoclast activity and hence a reduction in the release of tumour growth factors. However, it is also possible that bisphosphonates may have a direct effect on myeloma cells. In support of this we have demonstrated that bisphosphonates can decrease cell proliferation and induce apoptosis in human myeloma cells in vitro, and this review discusses the possibility that bisphosphonates may have not only an anti-resorptive action, but may also have a direct anti-tumour activity.

Antiproliferative effects of 9-beta-d-arabinofuranosyladenine in a mammalian cell line devoid of adenosine deaminase activity.

The effect of ara-A on cellular growth, DNA synthesis, and RNA synthesis, and RNA synthesis was measured in an established cell line (B-mix K-44/6) devoid of adenosine deaminase activity. Cells adapted to growth in a medium supplemented with horse serum provided an environment totally lacking adenosine deaminase activity whereas cultivation of cells in a medium supplemented with calf serum provided a system capable of deaminating ara-A to ara-H (half-life = 14 hours). Under deaminase-free conditions early log phase cells underwent 1.5 population doublings during 28 hours compared with 0.25 doublings in the presence of 37 micronM ara-A. When cells were grown in medium supplemented with calf serum the additionof 37 to 225 micronM ara-A resulted in a cessation of mitosis for periods of 5 to 30 hours respectively. Following this quiescent period growth resumed at the original rate. With 600 micronM ara-A mitosis was reversibly inhibited up to 35 hours after drug addition. The effects of ara-A on RNA and DNA synthesis were monitored by continuously or pulse labeling B-mix K-44/6 cells with [3H]-uridine or [3H]thymidine. Ara-A did not influence RNA synthesis as judged by labeled uridine incorporation. Under deaminase-free conditions, 5.4 micronM ara-A inhibited labeled thymidine incorporation by 50%. In the presence of the enzyme, approximately twice the ara-A concentration was required for the same inhibition; furthermore the initial inhibition was followed by a partial recovery in the rate of thymidine incorporation. Examination of thymidine incorporation. Examination of thymidine nucleotide pools during ara-A treatment revealed to changes in the labeling of dTMP, dTDP, and dTTP. Thus inhibition of [3H]thymidine incorporation by ara-A accurately reflected inhibition of DNA synthesis. We conclude that, in spite of an initial inhibition of DNA synthesis and mitosis by ara-A, B-mix K-44/6 cells recover from the inhibitory effects if the drug is removed either by a change in the culture medium or by metabolism to ara-H.

Kinetics of drug transport and concurrent metabolism in human cell cultures I: theory.

A method for evaluating the passive permeability and single-step metabolism of drugs in suspension cultures of mammalian cells was formulated assuming linear kinetics. It was assumed that the metabolizing enzymes are driven by endogenous substrates present in steady-state quantities. The presence of the drug in radiolabeled tracer quantities was assumed to cause only a small perturbation from the endogenous steady-state operating point. The time course solutions for the drug and its metabolite are given in terms of macroscopic constants, and their physical interpretations are given in terms of metabolic and transport parameters.