Comparison of immediate-release and controlled release carbidopa/levodopa in Parkinson's disease. A multicenter 5-year study. The CR First Study Group.

BACKGROUND: Motor response fluctuations and dyskinesias compromise long-term levodopa therapy in Parkinson's disease. Variations in plasma levodopa levels contribute to adverse reactions associated with chronic therapy. Therefore, sustained-release levodopa preparations may be associated with less motor fluctuations and a better outcome. We conducted a large, 5-year, multicenter study to address this hypothesis. METHODS: Six hundred and eighteen nonfluctuating patients with Parkinson's disease never exposed to levodopa therapy were randomized to (Sinemet CR 50/200) sustained-release or immediate-release (Sinemet 25/100) carbidopa/levodopa preparations in 35 centers worldwide. Dosage titration occurred over the 5 years of evaluations to maintain an optimal response. The primary endpoint, the 'event', was the presence of motor fluctuations, as defined by 20% 'off' time or 10% 'on' time with dyskinesias as recorded in the patient diary, or greater than or equal to a 50% positive response on the physician fluctuations questionnaire. Clinical rating scales, Nottingham Health Profile (NHP) and adverse reactions were also recorded. FINDINGS: During the 5 years of the study, both treatment groups responded extremely well to therapy. The incidence of all patients reaching the 'event' was low, approximately 20% by diary criteria and 16% by questionnaire definition, and there was no significant difference between the two treatment groups. Activities of daily living scores in the Unified Parkinson Disease Rating Scale (UPDRS) consistently favored the Sinemet CR treatment group and a number of the NHP scales also favored the CR group. Based upon the frequency of adverse experiences, and the overall low incidence of withdrawals, the two treatment groups demonstrated very similar safety profiles. The most common drug-related effect was nausea; seen in 20% of patients. Other drug-related effects were dizziness, insomnia, abdominal pain, dyskinesia, headache and depression. Drug-related withdrawals were less than 10% of all patients, primarily due to nervous/psychiatric complaints. INTERPRETATION: During a 5-year treatment period, control of parkinsonian symptoms was maintained by both immediate-release and sustained-release carbidopa/levodopa. Both treatment regimens were associated with a low incidence of motor fluctuations and dyskinesias. There was a statistically significant difference (p < 0.05) in activities of daily living as measured by the UPDRS in favor of Sinemet CR.

Expansion of lymphokine-activated killer cells for clinical use utilizing a novel culture device.

Two major problems encountered in the application of lymphokine-activated killer (LAK) cell therapy in man are the massive culture volumes required for LAK cell induction and the paucity of LAK cells available for administration (human doses are less than or equal to 10% of effective murine LAK cell doses). We have, therefore, developed and tested a plastic porous culture device, Sclair plastic bags (E.I. DuPont De Nemours Co.), that can be utilized at virtually any volume and does not require rotation for optimal use. Normal or patient lymphocytes were cultured in the device or in plastic 16 mm wells at 1-20 X 10(6)/ml RPMI 10% human sera with 1500 pM interleukin-2 for 4 days: LAK cell activity did not decline despite high cell densities. The device was equal to the 16 mm wells in induction of normal donor and patient LAK cell activity when either autologous fresh tumor or Raji targets were used. In a non-therapeutic clinical evaluation we isolated and stored in liquid nitrogen autologous tumor cells from 11 patients with cancer. 2-6 weeks post-operatively lymphocytes and mononuclear cells from these patients and paired normal donors were obtained and LAK cells were induced in Sclair bags or standard culture wells. Autologous patient LAK cell activity and normal donor LAK cell activity against patient's tumor cells were equivalent in the Sclair culture device and culture well system. Lymphocyte recovery and [3H]thymidine incorporation were also similar. Subsequently, we developed an expansion scheme utilizing the device in which cell density was maintained at optimal levels by changing media and reducing cell concentration after 6, 10 and 14 days of culture. We were able to expand LAK cell number 5-10-fold with no loss of LAK cell activity in this time frame utilizing both normal and patient cells. In this system plasma and sera were equivalent in their capacity to support LAK cell expansion but less than 10% plasma or sera supported suboptimal activation. Thus, we have developed a practical system to augment the number of LAK cells available for human LAK cell therapy and simultaneously reduce the complexity and volume of the induction system.

A procedure for the statistical evaluation of Ames Salmonella assay results. Comparison of results among 4 laboratories.

Ames Salmonella test data collected in our laboratory and 3 National Cancer Institute contract laboratories were analyzed to study the distribution of experimental errors associated with the test. It is shown that the Poisson distribution is not appropriate, and that the power transformation model Y = (revertants/plate)lambda, with lambda = 0.2 as estimated by the methods of Box and Cox, produced a measurement scale on which the experimental errors could be adequately described by a normal (Gaussian) distribution with a constant variance. The modeling procedure enables one to properly use analysis of variance, regression analysis, and Student's t test to analyze Ames Salmonella test results, and well-known statistical quality control procedures to monitor laboratory performance. The method detects weak mutagenic activity and measures the amount and uncertainty of the increase in revertants/plate. The development of the power transformation model is discussed and examples of its use in the interpretation of Ames Salmonella assay results are included.

The use of cytogenetic monitoring in the workplace: a position paper of the American Industrial Health Council.

Although tests to determine cytogenetic effects can be used to identify workers who have been exposed to certain agents, uncertainties remain. Tests to determine cytogenetic effects have not gained wide acceptance for routine monitoring in the workplace because of questions concerning their general applicability to detect exposure to a wide variety of agents and because of the lack of data showing a correlation between cytogenetic effects as detected in humans and adverse health effects. Unless those issues are resolved, the measurement of cytogenetic effects for workplace monitoring purposes should be regarded as experimental and such measurement is not likely to become a routine workplace exposure monitoring technique.

A statistical method for analysis of mouse lymphoma L5178Y cell TK locus forward mutation assay. Comparison of results among three laboratories.

Analysis of data from our laboratory and that of two National Cancer Institute contractor laboratories indicate the random variation in the results of the mouse lymphoma L5178Y cell TK locus mutation assay can be adequately described by a lognormal distribution. This indicates that transformation of mutant frequencies to logarithms enables one to properly use well-known statistical techniques such as analysis of variance, regression analysis, and Student's t-test for the interpretation of data from this assay. The consistency of the lognormal distribution among laboratories is demonstrated. Three examples which illustrate the mechanics and interpretation of the proposed methodology are included. It is concluded that the method is effective in identifying weak mutagens as well as enabling the user to compute the uncertainty associated with an observed increase in mutagenic activity.

Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells.

Mutagenesis data collected in the mammalian cell CHO/HGPRT assay were analyzed to study the distribution of the experimental errors associated with the test. The data neither followed the widely assumed Poisson distribution nor satisfied the usual statistical assumptions of normality and homogeneous variance of experimental errors. We transformed the data by using the power formula Y = (X + A) gamma where X is the observed mutation frequency, Y is the transformed frequency, and A and gamma are constants determined by the procedure of Box and Cox. Setting A = 1 and gamma = 0.15 we produced transformed values for which the assumptions of homogeneous variance and normal distribution were satisfied. This transformation enables one to properly use Student's t-test and dose-response analysis of variance to analyze CHO/HGPRT results. The experimental design for CHO/HGPRT mutagenesis assays is also discussed.

Control of argininosuccinate synthetase by arginine in human lymphoblasts.

Human lymphoblasts in long-term culture have the enzyme activities necessary to convert citrulline to arginine: argininosuccinate synthetase and argininosuccinate lyase. Upon transfer from arginine-supplemented to citrulline-supplemented medium, lymphoblasts exhibit a lag period before resuming exponential growth. During this lag the specific activity of argininosuccinate synthetase increases an average of 60-fold. Argininosuccinate lyase activity remains unchanged. If normal lymphoblasts are starved in arginine-deficient medium without citrulline or if argininosuccinate lyase--deficient lymphoblasts are transferred to citrulline-containing medium, argininosuccinate synthetase activity increases linearly for several days and reaches even higher levels. Cycloheximide blocks the increase in enzyme activity. Cells grown in citrulline medium and pulse labeled with 35S-methionine incorporate more 35S-methionine into argininosuccinate synthetase protein than cells grown in arginine; the rate of disappearance of radioactively labeled enzyme is the same in citrulline- and arginine-grown cells. Arginine or a closely related metabolite thus appears to repress the synthesis of argininosuccinate synthetase of human lymphoblasts in culture.

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.

Maturation of ribosomal RNA in stringent and relaxed bacteria.

Experiments with relaxed and stringent strains of E. coli confirm that rRNA synthesis continues in both during amino acid starvation. rRNA species produced are exclusively in their precursor configurations and vulnerable to nuclease attack.

L-arabinose isomerase formation in a conditional mutant of gene araA of Escherichia coli B-r.

A temperature-sensitive mutant of Escherichia coli in which the synthesis of l-arabinose isomerase is blocked during growth at 42 C was found to possess the following properties. (i) The mutation occurred in the structural gene for the isomerase, gene araA. (ii) During growth at elevated temperatures the mutant accumulates a product which is a precursor to the active enzyme. (iii) The precursor produced at 42 C is slowly converted to active enzyme at 28 C in the absence of protein and ribonucleic acid synthesis. It is concluded that the mutation results in a change in the structure of isomerase which causes formation of active enzyme to be thermolabile at a step beyond the level of translation.

Control of nucleotide metabolism and ribosomal ribonucleic acid synthesis during nitrogen starvation of Escherichia coli.

Ribosomal ribonucleic acid (RNA) synthesis and ribonucleoside triphosphate metabolism were studied in cultures of Escherichia coli subjected to starvation for inorganic nitrogen. In a strain that was under stringent control, a 50-fold reduction in the formation of both 16S and 23S RNA was accompanied by a severe restriction on nucleotide biosynthesis. These inhibitions were relieved in part by incubating the starved cells with amino acids. This result suggests that regulation by the functional RNA control (RC) gene is involved in the effect. This suggestion was confirmed by showing that the effector of the stringent response, guanosine-5'-diphosphate-2'- or 3'-diphosphate ((pp)G(pp)), accumulated at the onset of starvation and disappeared immediately when the amino acids were added. Ribosomal RNA synthesis was severely restricted and the same nucleotide, (pp)G(pp), accumulated at the onset of nitrogen starvation of a relaxed mutant too. These findings suggest that a control mechanism other than the one provided by the functional rel gene might operate to regulate RNA synthesis and that this mechanism is expressed through the synthesis of (pp)G(pp).

Control of expression of the L-arabinose operon in temperature-sensitive mutants of gene araC in Escherichia coli B-r.

Expression of the l-arabinose operon in Escherichia coli B/r is dependent on the temperature of growth of the araC mutants reported in this paper. Analysis of these temperature-sensitive regulatory mutants indicates that both repressor and activator activities are thermolabile. The simplest model to explain the manner in which the operon is controlled is one suggesting that the regulatory gene, araC, codes for a protein which upon synthesis acts as a repressor molecule and prevents operon function. When inducer is added, the repressor undergoes a conformational shift and becomes an activator which switches on enzyme synthesis, provided the repressor concentration is reduced to a sufficiently low level in the cell. These data lend strong support to the model that both activities are the result of the same gene product.