Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Psychiatry under pressure: reflections on psychiatry's drift towards a reductionist biomedical conception of mental illness.

We argue that contemporary psychiatry adopts a defensive strategy vis-à-vis various external sources of pressure. We will identify two of these sources--the plea for individual autonomy and the idea of Managed Care--and explain how they have promoted a strict biomedical conception of disease. The demand for objectivity, however, does not take into account the complexity of mental illness. It ignores that the psychiatrist's profession is essentially characterized by fragility: fluctuating between scientific reduction and the irreducible complexity of reality. Therefore, the psychiatrist is not in need of hard and fast rules, but of judgment. At the end, we suggest that philosophy could inject some healthy uncertainty within psychiatry in order to restore its fragile identity. Our examples are drawn from the Dutch situation but we are confident that they apply to other countries as well.

Internal jugular vein thrombosis following in-vitro fertilization in a woman with protein S deficiency and heterozygosity for the prothrombin 3' UTR mutation, despite anticoagulation with heparin.

We report the case of a 31-year-old woman with protein S deficiency and heterozygosity for the prothrombin 3' UTR mutation who developed an internal jugular vein thrombosis despite therapeutic anticoagulation with a low molecular weight heparin, following in-vitro fertilization. This case indicates that the stimulus to thrombosis in such women is intense and can occur despite apparent therapeutic anticoagulation. Close attention should, therefore, be paid to any women with a personal or family history of thrombosis and the potential thrombotic risks associated with assisted conception must be discussed.

Working with community mental health professionals: a survey among general practitioners.

Links between general practitioners and mental health professionals, such as counsellors, psychiatrists, community psychiatric nurses, clinical psychologists and social workers, are increasing in number and type. The aim of this survey was to elicit general practitioners' attitudes to these workers, comparing those with a link with a mental health worker and those without. General practitioners in two district health authorities were surveyed and a response rate of 70% was obtained. General practitioners linked to a mental health professional were more likely to have made a referral to that service in the previous three months and, on the whole, were more satisfied with that service. The commonest problem reported by respondents was the length of waiting lists. Regarding liaison with social workers, inadequate feedback and difficulty with contact were the problems mentioned most by doctors. A number of general practitioners expressed a desire for closer contact with all these mental health services. While caution is required in ascribing causality to these relationships, it is clear that a closer working relationship between general practitioners and mental health workers is productive and is valued by general practitioners. The challenge for policy makers is to structure mental health provision in such a way that more general practitioners are able to benefit than at present.

A survey of links between mental health professionals and general practice in six district health authorities.

The aim of this study was to obtain an estimate of the extent to which collaborative schemes exist between general practice and mental health professionals and to assess the influence of practice size and district on these schemes. A questionnaire asking about such links was sent to each general practice in six randomly selected health districts in England. The response rate was 75%. Half of the 261 responding practices had a link with a community psychiatric nurse, 21% with a social worker, 17% with a counsellor, 15% with a clinical psychologist and 16% with a psychiatrist. Practices with more general practitioners were significantly more likely to have a link with a counsellor, after allowing for marked differences between the sizes of practices in the different districts. There was a tendency for some practices to have many links, while others had few. This poses questions about the efficiency and equity of collaborative schemes in primary care. Further research is required to investigate the quality of these links and the extent to which they serve the interests of the patient.

Pharmacokinetics of 2',3'-dideoxyinosine in patients with severe human immunodeficiency infection. II. The effects of different oral formulations and the presence of other medications.

2',3'-Dideoxyinosine (ddI) has shown activity against human immunodeficiency virus in phase I clinical trials. The drug is rapidly degraded by acid, however, thus raising questions as to the efficiency and reproducibility of its absorption after oral administration. This investigation studies the bioavailability of several oral dosage forms of ddI. When ddI was given to fasting patients as an oral solution with antacid, the bioavailability was 41% +/- 7% (mean +/- SEM). However, when given as buffered tablets, the bioavailability was considerably less (25% +/- 5%). The bioavailability increased slightly when the tablets were given with supplemental antacid (36% +/- 6%). Two enteric-coated preparations had reasonable bioavailability (36% +/- 5% and 26% +/- 5%), but the peak plasma level was much lower and occurred at a much later time than with the oral solution. When ddI was given as a premeasured powder containing sucrose and buffer to be reconstituted by the patient (the "sachet" preparation), the bioavailability was 29% +/- 6%. This was similar to that of the oral solution for this particular group of patients (30% +/- 7%). However, the bioavailability of the sachet was only 17% +/- 4% when administered with food. When the sachet was given to patients receiving ranitidine, no consistent change in bioavailability was noted. Also, no change in ddI pharmacokinetics was noted in patients receiving ganciclovir.

Quantitative analysis of HIV-1 proviral DNA in peripheral blood mononuclear cells from patients with AIDS or ARC: decrease of proviral DNA content following treatment with 2',3'-dideoxyinosine (ddI).

A rapid and simple method for determining the proviral DNA content in peripheral blood mononuclear cells (PBM) from patients with human immunodeficiency virus type 1 (HIV-1) infection was established by using the polymerase chain reaction (PCR) technique. We found that the majority of HIV proviral DNA copies detectable in unfractionated PBM resided in T cells, while B cells/monocytes contained lesser amounts of HIV DNA (93.9 +/- 3.5% for T cells vs. 6.1 +/- 3.5% for B cells/monocytes: p less than 0.05). When we compared the amount of HIV proviral DNA in PBM from 13 patients with AIDS or AIDS-related complex (ARC) before and during antiretroviral therapy with 2',3'-dideoxyinosine (ddI) which was given as an escalating dose in a Phase I clinical study, a significant decrease was observed in 9 of 12 evaluable patients receiving the drug for 8 to 14 weeks (p less than 0.02). The decrease appeared more pronounced in patients receiving relatively high doses of the drug. These data suggest that the quantitation of HIV viral DNA in PBM by PCR is feasible and may theoretically contribute to an overall monitoring of patients receiving experimental therapy. However, larger studies will be required to determine the sensitivity and specificity of this assay and further longitudinal studies will be essential.

New drug therapy for patients with HIV. Nursing implications in the administration of 2',3'-dideoxyinosine (ddI).

2',3'-Dideoxyinosine (ddI) is a dideoxynucleoside currently in Phase I, II, and III trials for antiretroviral therapy. It has been shown to cause objective and subjective improvement in people with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC). This drug, as with any drug, is not without toxicity. Through thorough patient education and clinical evaluation, incidence of these toxicities may be lessened or avoided.

Long-term toxicity/activity profile of 2',3'-dideoxyinosine in AIDS or AIDS-related complex.

To evaluate the long-term toxicity and activity profile of 2',3'-dideoxyinosine (ddI), a potent inhibitor of human immunodeficiency virus (HIV) replication, in vitro. 58 patients with AIDS or AIDS-related complex were studied with additional reference to the effect of previous treatment with zidovudine, and the effect of ddI on HIV-induced cognitive dysfunction. Doses above 9.6 mg/kg per day of ddI were frequently associated with toxicity (peripheral neuropathy, pancreatitis, or hepatitis). Doses of 9.6 mg/kg per day or below were well tolerated for up to 21 months. A subset of patients receiving 3.2-9.6 mg/kg per day of ddI had long-term immunological improvement and reduction of serum HIV p24 antigen. Immunological changes were especially seen in patients who had little previous zidovudine therapy. 5 patients with HIV-induced cognitive impairment improved with ddI. Thus, ddI may have anti-HIV activity at doses which are tolerated for long-term therapy, although pancreatitis could be a life-threatening complication.

The National Cancer Institute phase I study of 2',3'-dideoxyinosine administration in adults with AIDS or AIDS-related complex: analysis of activity and toxicity profiles.

2',3'-Dideoxyinosine (didanosine; ddI) was administered to 37 adults with AIDS or AIDS-related complex in an escalating-dose phase I study. Groups of three or four patients received intravenous dosages of 0.4 mg/(kg.d) to 25.6 mg/(kg.d) divided into two or three daily doses for 2 weeks, followed by oral ddI at twice the intravenous dosages. When given with antacids, ddI was well absorbed by the oral route and penetrated into the cerebrospinal fluid. The patients had an increase in mean number of CD4+ cells from 114/mm3 at entry to 161/mm3 at week 6 (P = .00004). They also had an increase in the CD4+/CD8+ ratio and in total number of lymphocytes. Sixteen of 18 evaluable patients had a decrease in levels of human immunodeficiency virus p24 antigen by week 6 (P = .0034). Many patients reported increased energy and appetite and gained weight. Dose-limiting toxicities at high dosages were painful peripheral neuropathy and sporadic pancreatitis. However, dosages up to 9.6 mg/(kg.d) have been tolerated in patients for 11-14 months. Thus, ddI has activity against human immunodeficiency virus at dosages that can be tolerated for approximately 1 year. However, life-threatening pancreatitis is a possible complication even at low dosages, and the best ways to manage and avoid adverse effects are still under study.

Pharmacokinetics of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine in patients with severe human immunodeficiency virus infection.

This article describes the pharmacokinetics of 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) as determined during phase I clinical trials in patients with acquired immunodeficiency syndrome and acquired immunodeficiency syndrome-related complex. Drug levels were determined by HPLC in plasma, cerebrospinal fluid, and urine after administration of the drugs either intravenously or as an oral liquid given with antacid. ddA was metabolized rapidly and quantitatively to ddI to such an extent that ddA was undetectable in the plasma even during continuous intravenous administration of ddA. The plasma kinetics of ddI were generally monoexponential and were characterized by a half-life of 38 minutes. This probably does not accurately reflect the kinetics of the active species of ddI, which appears to be 2',3'-dideoxyadenosine triphosphate, formed intracellularly. Oral bioavailability was 38% for oral liquid given with antacid. The total body clearance averaged 1.00 L/kg/hr, with a volume of distribution of 1.01 L/kg. Approximately 36% of the intravenous dose could be recovered unchanged in the urine. The level of ddI in the cerebrospinal fluid 1 hour after drug infusion averaged 21% of that of the simultaneous plasma level. It is concluded that ddI has pharmacokinetic properties that are amenable to its clinical use.

Phase I and pharmacokinetic study of arabinofuranosyl-5-azacytosine (fazarabine, NSC 281272).

A Phase I clinical trial of 1-beta-D-arabinofuranosyl-5-azacytosine (ara-AC or fazarabine) given as a 72-h continuous infusion on a 21-day cycle was conducted in 27 adult patients with refractory cancer. The major toxicity was reversible granulocytopenia and thrombocytopenia. Dose-limiting toxicity was observed at a dose rate of 1.96 mg/m2/h in which Grade IV leukopenia (WBC less than 1,000/mm3) occurred in 4 of 11 patients and Grade IV thrombocytopenia (platelets less than 25,000/mm3) occurred in 3 of 11 patients. Plasma steady-state levels ranged from 0.13 to 0.6 microM for doses of 1.25 to 5.94 mg/m2/h. Mean total body clearance was 647 ml/min/m2. Minor clinical responses were seen in one patient with testicular cancer, one patient with colon cancer, one patient with breast cancer, and one patient with acute nonlymphocytic leukemia. Another patient with adenocarcinoma of unknown primary had stable disease during 13 cycles of therapy. Based on the results of this study, the recommended dose for Phase II studies of 1-beta-D-arabinofuranosyl-5-azacytosine administered as a 72-h continuous infusion is 2.0 mg/m2/h (48 mg/m2/day).

Initial clinical experience with dideoxynucleosides as single agents and in combination therapy.

Several dideoxynucleosides, including 3'-azido-2',3'-dideoxythymidine (zidovudine, azidothymidine, AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyinosine (ddI), have been shown to be potent inhibitors of human immunodeficiency virus (HIV) replication in human T cells and macrophages. These compounds undergo anabolic phosphorylation within target cells to a 3'-triphosphate moiety; as triphosphates, they act at the level of HIV DNA polymerase (reverse transcriptase). AZT has been shown to reduce the morbidity and mortality of patients with severe HIV infection and to at least temporarily ameliorate certain cases of HIV-induced dementia. In phase 1 studies, ddC and ddI have been shown to induce immunologic and virologic improvements in patients with AIDS or related disorders; phase 2 studies of ddC and ddI are underway. The use of these drugs can be associated with toxicity. AZT can cause bone marrow toxicity or myositis with prolonged use, ddC can cause peripheral neuropathy at high doses, and ddI can cause sporadic pancreatitis and peripheral neuropathy at high doses. For each compound, however, a therapeutic window exists in which an anti-HIV effect can be attained without short-term toxicity in most patients. Dose-intensity appears to be an important determinant of the toxicity of dideoxynucleosides. Studies are underway to explore how the therapeutic profiles of these compounds may be enhanced by attention to scheduling or through the use of combination therapy.

Alteration of zidovudine pharmacokinetics by probenecid in patients with AIDS or AIDS-related complex.

The anti-human immunodeficiency virus drug zidovudine is metabolized extensively in human beings to the 5'-glucuronide (GAZT) and is cleared rapidly, resulting in a short half-life and the need for frequent dosing. This study explores whether probenecid, which is also metabolized by glucuronidation, reduces zidovudine clearance when zidovudine is administered orally to patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). The mean zidovudine plasma levels were significantly higher after concurrent administration of probenecid than in its absence, resulting in a twofold increase in the mean AUC, a corresponding decline in the apparent total clearance, and a prolongation in the mean half-life. Similar alterations were observed in GAZT disposition. There was a marked reduction in the urinary excretion ratio of GAZT to zidovudine and a decline in the renal clearance of GAZT after probenecid coadministration. Probenecid inhibits zidovudine glucuronidation and renal excretion of GAZT.

In vivo activity against HIV and favorable toxicity profile of 2',3'-dideoxyinosine.

The purine analog 2',3'-dideoxyinosine (ddI), which has anti-retroviral activity in vitro was administered for up to 42 weeks to 26 patients with acquired immunodeficiency syndrome (AIDS) or severe AIDS-related complex (ARC). Ten of these individuals were AZT-intolerant. Eight dose regimens were studied. The drug was orally bioavailable and penetrated into the cerebrospinal fluid (CSF). Comparatively little evidence of an effect against human immunodeficiency virus (HIV) was seen at the lowest four doses. However, patients in the four highest dose groups (ddI at 1.6 milligrams per kilogram intravenously and then greater than or equal to 3.2 milligrams per kilogram orally at least every 12 hours or higher) had increases in their circulating CD4+ T cells (P less than 0.0005), increased CD4/CD8 T cell ratios (P less than 0.01), and, where evaluable, more than an 80% decrease in serum HIV p24 antigen (P less than 0.05). The patients also had evidence of improved immunologic function, had reduced viremic symptomatology, and gained a mean of 1.6 kilogram with these comparatively infrequent dosing schedules (every 8 or 12 hours). The most notable adverse effects directly attributable to ddI administration at the doses used in this study included increases in serum uric acid (due to hypoxanthine release) and mild headaches and insomnia. These results suggest that serious short-term toxicity at therapeutic doses is not an inherent feature in the profile of agents with clinical anti-HIV activity. Further controlled studies to define the safety and efficacy of this agent may be worth considering.

Treatment of the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex with a regimen of 3'-azido-2',3'-dideoxythymidine (azidothymidine or zidovudine) and acyclovir. A pilot study.

On the basis of observation that acyclovir potentiates the in-vitro antiviral activity of 3-azido-2',3'-dideoxythymidine (also known as azidothymidine or zidovudine) against human immunodeficiency virus (HIV), we administered a regimen of azidothymidine and acyclovir to eight patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. An oral regimen of 100 mg of azidothymidine and 800 mg of acyclovir every 4 hours was in general well tolerated, with the principal toxicity being megaloblastic erythroid changes. The pharmacokinetics of the two drugs were independent of each other. Six patients received the drug combination for at least 10 weeks; all had increased numbers of T4+ lymphocytes (P = 0.028), and two of three assessable patients had reversal of anergy. Two patients tested positive for serum HIV p24 antigen at entry, but became negative with treatment. Data for this small group suggest that this drug combination can be tolerated in patients with severe HIV infections; this study can be used as a basis for larger studies of this drug combination.

Long-term administration of 3'-azido-2',3'-dideoxythymidine to patients with AIDS-related neurological disease.

3'-Azido-2',3'-dideoxythymidine (AZT) has been administered to 7 patients with human immunodeficiency virus-associated neurological disease: 3 with dementia, 2 with peripheral neuropathy, 1 with dementia and peripheral neuropathy, and 1 with T-10 paraplegia. Six of the patients showed improvement in their neurological dysfunction on being administered AZT, as assessed by clinical evaluation, neuropsychological testing, nerve conduction studies, and/or positron emission tomographic scans. Three of these 6 patients showed sustained improvement 5 to 18 months after the initiation of AZT therapy. These results suggest that certain human immunodeficiency virus-associated neurological abnormalities are at least partially reversible following the administration of antiretroviral therapy and provide a rationale for further studies using antiretroviral chemotherapy.