Influence of the Plasmodium falciparum P-glycoprotein homologue 1 (pfmdr1 gene product) on the antimalarial action of cyclosporin.

BACKGROUND AND OBJECTIVES: The immunosuppressant cyclosporin A and a number of other cyclosporins have potent and selective antimalarial activity. Their exact mechanism of antimalarial action is unknown but the structure-activity relationships for malarial parasite inhibition and immunosuppression differ markedly. The 3'-keto derivative of cyclosporin D (valspodar) is particularly potent against the human malarial parasite Plasmodium falciparum in culture but causes negligible immunosuppression. Multidrug resistance in mammalian cancer cells, the result of overproduction of the P-glycoprotein, can be reversed by certain cyclosporins, particularly valspodar. We therefore investigated the possibility that the antimalarial target of cyclosporin might be a P-glycoprotein homologue. P. falciparum P-glycoprotein homologue 1 (Pgh1; the pfmdr1 gene product) is located in the digestive vacuole (DV) membrane of the parasite. Its function is unknown but it modulates the susceptibility of parasites to quinolines and related antimalarial drugs, including quinine, mefloquine, halofantrine and chloroquine, and to artemisinin. METHODS AND RESULTS: Here we demonstrate that (i) sequence polymorphisms in pfmdr1 altered the susceptibility of parasites to cyclosporin A and (ii) pfmdr1-overexpressing strains were slightly less susceptible to the drug. Furthermore, we found synergistic antimalarial interactions between cyclosporin A and quinine, mefloquine or halofantrine and antagonism between cyclosporin A and chloroquine. However, we were unable to detect a direct interaction between cyclosporin and Pgh1. CONCLUSIONS: The amino acid sequence and copy number of Pgh1 may influence cyclosporin susceptibility as a result of a direct interaction between the drug and the protein, or via indirect effects on the physiology of the DV.

Analysis of antimalarial synergy between bestatin and endoprotease inhibitors using statistical response-surface modelling.

The pathway of hemoglobin degradation by erythrocytic stages of the human malarial parasite Plasmodium falciparum involves initial cleavages of globin chains, catalyzed by several endoproteases, followed by liberation of amino acids from the resulting peptides, probably by aminopeptidases. This pathway is considered a promising chemotherapeutic target, especially in view of the antimalarial synergy observed between inhibitors of aspartyl and cysteine endoproteases. We have applied response-surface modelling to assess antimalarial interactions between endoprotease and aminopeptidase inhibitors using cultured P. falciparum parasites. The synergies observed were consistent with a combined role of endoproteases and aminopeptidases in hemoglobin catabolism in this organism. As synergies between antimicrobial agents are often inferred without proper statistical analysis, the model used may be widely applied in studies of antimicrobial drug interactions.

Genetic parameters of test day milk yields of Holstein cows

No presente estudo foram utilizados 17.968 registros de produçäo de leite, referentes a 2.130 primeiras lactaçöes de vacas da raça Holandesa, paridas nos anos de 1988 a 1991, filhas de 136 touros e controladas pela Associaçäo Brasileira de Criadores (ABC). Os dados foram distribuídos em dez sub-arquivos de acordo com o número do controle (M1 e M10). As características estudadas foram: produçäo de leite no dia do controle (M) e produçäo aos 305 dias de lactaçäo (M305), as quais foram ajustadas para os seguintes fatores de variaçäo: idade da vaca ao parto em classes, intervalo parto-primeiro controle e subclasses de rebanho-ano-estaçäo de parto. Os componentes de (co)variância foram obtidos a partir de análises com duas características utilizando-se o método REML, sob um modelo de touro. As estimativas de herdabilidade para as M variaram de 0,04 a 0,32 sendo os maiores valores encontrados na segunda fase da lactaçäo (M5 a M7), enquanto que para a M305 este valor foi de 0,32. As correlaçöes genéticas entre as M e a M305 variaram de 0,78 a 1,00. Os resultados sugerem que as M podem ser utilizadas em substituiçäo à M305 nas avaliaçöes genéticas dos animais, visto que as herdabilidades no meio da lactaçäo foram semelhantes à encontrada para a M305. Além disso, a seleçäo antecipada pode levar a uma reduçäo no intervalo de geraçöes.

A regulatory perspective on pharmacokinetic/pharmacodynamic modelling.

We present an integrated summary from statistical and pharmacological perspectives of pharmacokinetic/pharmacodynamic (PK/PD) modelling and its use in drug development and regulation for guiding appropriate dosing. An overview of the technical aspects of PK/PD modelling describes how structural models are constructed and refined using pharmacokinetic and pharmacodynamic principles and how random effects models are used to account for individual differences in desired (and undesired) responses due to patient characteristics. Lastly, we describe applications of PK/PD modelling for the purposes of drug labelling, for resolving a safety concern, and for improving therapeutic monitoring of anaesthetic depth during surgery.

Evaluation of designs for clinical trials of neuroprotective agents in head injury. European Brain Injury Consortium.

In a study involving the statistical modeling of potential head injury trials, we explore approaches to trial design that could enhance their power to detect treatment-related effects on clinical outcome. The study uses a survey organized by the European Brain Injury Consortium of over 1,000 head-injured patients to characterize the population from which trial participants can be selected. A variety of models are postulated for the effects of "neuroprotective" treatment on outcome, and their interaction with a range of strategies for targeting patients for inclusion in the trial is evaluated. A very simple strategy of targeting patients with an intermediate prognosis was found to allow a reduction in sample size by 30%, with no reduction in statistical power. This paper illustrates an important methodology for studying the characteristics of competing trial designs.

An approach to the validation of markers for use in AIDS clinical trials.

Dr. Mildvan and coauthors have thoroughly reviewed and documented what is known about the validation of surrogate markers for use in clinical trials. They have proposed a classification system based on the usefulness of available immunologic and virological assays as measures of prognosis, drug activity, and therapeutic efficacy. The latter, a type II marker in the proposed classification, should estimate the proportion of treatment effect explained by change in the marker induced by therapy and, if complete, can substitute for clinical endpoints. HIV clinical trialists have had a long-standing interest in using surrogates for clinical endpoints to facilitate conduct of experimental protocols and to decrease the time and effort required to develop new treatment strategies. The approach outlined in this review by experienced clinicians, biostatisticians, and immunologists provides a framework to evaluate currently available and potential surrogate markers.

A direct, general approach based on isobolograms for assessing the joint action of drugs in pre-clinical experiments.

Pharmacologists and other biologists frequently use methods based on the interpretation of isobolograms to quantify the extent of synergy or antagonism between drugs used in combination in pre-clinical studies. Most methods have been unsatisfactory from a statistical viewpoint, many because they have relied solely on visual evaluation, others because the methods have not taken into account the variability of the measurements. We describe a direct approach for quantifying the joint potency of two drugs, a central feature being the use of simple isobole models that lead directly to response surface models for the expected experimental outcomes. The approach is general in the sense that one can use it for discrete or continuous responses, different underlying probability distributions, linear or non-linear dose-response functions of the drugs used singly, and a variety of experimental designs. Our approach extends the suggestions made by Hewlett for measuring the joint potency of drugs, and is similar in spirit to the approaches proposed by Greco et al. and Weinstein et al. We describe the analysis of data from an in vitro experiment conducted to evaluate the efficacy of the antiviral drugs AZT and ddI used in combination.

A case-control interview study of breast cancer among Japanese A-bomb survivors. I. Main effects.

Women with breast cancer (cases = 196) and without the disease (controls = 566), selected from the Life Span Study sample of A-bomb survivors and nonexposed residents of Hiroshima and Nagasaki, Japan, and matched on age at the time of the bombings, city, and estimated radiation dose, were interviewed about reproductive and medical history. A primary purpose of the study was to identify strong breast cancer risk factors that could be investigated further for possible interactions with radiation dose. As expected, age at first full-term pregnancy was strongly and positively related to risk. Inverse associations were observed with number of births and total, cumulative period of breast feeding, even after adjustment for age at first full-term pregnancy. Histories of treatment for dysmenorrhea and for uterine or ovarian surgery were associated positively and significantly with risk at ages 55 or older, a finding that requires additional study. Other factors related to risk at older ages were the Quetelet index (weight [kg]/height [cm]2) at age 50, history of thyroid disease, and hypertension. Neither age at menarche nor age at menopause was associated significantly with risk. Subjects appeared to be poorly informed about history of breast cancer or other cancer in themselves or in their close relatives; this finding suggests that innovative strategies may be required when studying familial cancer patterns in Japanese populations.

A case-control interview study of breast cancer among Japanese A-bomb survivors. II. Interactions with radiation dose.

Three breast cancer risk factors were evaluated in terms of their interactions with radiation dose in a case-control interview study of Japanese A-bomb survivors. Cases and controls were matched on age at the time of the bombings and radiation dose, and dose-related risk was estimated from cohort rather than case-control data. Each factor--age at first full-term pregnancy, number of deliveries, and cumulative lactation period summed over births--conformed reasonably well to a multiplicative interaction model with radiation dose (the additive interactive model, in which the absolute excess risk associated with a factor is assumed to be independent of radiation dose, was rejected). An important implication of the finding is that early age at first full-term pregnancy, multiple births, and lengthy cumulative lactation are all protective against radiation-related, as well as baseline, breast cancer. Analyses by age at exposure to radiation suggest that, among women exposed to radiation in childhood or adolescence, a first full-term pregnancy at an early age following exposure may be protective against radiation-related risk.

Endogenous interferon and triglyceride concentrations to assess response to zidovudine in AIDS and advanced AIDS-related complex.

To improve evaluation of new antiretroviral drugs in the acquired immunodeficiency syndrome (AIDS), sensitive biological markers that accurately predict response to treatment are needed. Two possible markers are endogenous interferon (E-IFN), which is a cytokine involved in the pathophysiology of AIDS, and serum triglycerides (TG), which are raised in patients with AIDS, possibly reflecting enhanced cytokine activity. E-IFN, TG, body-mass index, CD4 count, and HIV p24 were measured in 19 patients (15 with AIDS, 4 with AIDS-related complex), who were part of the phase II licensing trial of zidovudine (ZDV). 10 received ZDV and 9 received placebo. Rapid, significant, and sustained declines from initial values in E-IFN and TG concentrations were observed in ZDV patients but not in placebo patients. Baseline values of E-IFN and TG concentrations after 4 months on ZDV treatment were both important contributors to long-term survival. The findings suggest that these indicators of abnormal cytokine expression may be useful measures of not only disease severity but also efficacy of antiretroviral therapy in AIDS.

Human immunodeficiency virus infection of human lymph nodes in the SCID-hu mouse.

The SCID-hu mouse is a small animal in which human hematolymphoid organs can be engrafted and maintained in vivo. In this study, parameters are described for reproducible infection of SCID-hu mice after i.v. inoculation. Infection was found to be dependent upon the time after inoculation, the virus isolate, the titer of virus, and the human target organ implanted into the mouse. Ten to 14 days after the i.v. administration of HIV isolates derived freshly from patients (e.g., JR-CSF, JR-FL, SM), 100% of engrafted human lymph nodes in SCID-hu mice were infected; greater than 95% of these animals were also viremic. Implants of human thymus or connective tissue, as well as the endogenous murine hematolymphoid organs, were not infected. As demonstrated by a combination of in situ hybridization and immunohistochemistry, both T-lymphoid and myelomonocytic lineage cells were infected in this system. HIV isolates that have been adapted to growth in vitro (e.g., HTLV-IIIb) were not infectious. When either 3'-azido-3'-deoxythymidine (AZT) or 2',3'-dideoxyinosine (ddIno) was administered to SCID-hu mice before HIV infection, the animals were protected in dose ranges similar to those used in man. This animal model may now be used as an efficient intermediate step between the lab and the clinic to study the infectious process in vivo and to best select efficacious antiviral compounds against HIV.

Chromosome aberrations in relation to radiation dose following partial-body exposures in three populations.

Structural chromosome aberrations were evaluated in peripheral blood samples obtained from three populations exposed to partial-body irradiation. These included 143 persons who received radiotherapy for enlarged thymus glands during infancy and 50 sibling controls; 79 persons irradiated for enlarged tonsils and 81 persons surgically treated for the same condition during childhood; and 77 women frequently exposed as young adults to fluoroscopic chest X rays during lung collapse treatment for tuberculosis (TB) and 66 women of similar ages treated for TB with other therapies. Radiation exposures occurred 30 and more years before blood was drawn. Doses to active bone marrow averaged over the entire body were 21, 6, and 14 cGy for the exposed thymic, tonsil, and TB subjects, respectively. Two hundred metaphases were scored for each subject, and the frequencies of symmetrical (stable) and asymmetrical (unstable) chromosome aberrations were quantified in 97,200 metaphases. Cells with stable aberrations were detected with greater frequency in the irradiated subjects compared with nonirradiated subjects in all three populations, and an overall test for an association between stable aberrations and partial-body ionizing radiation was highly significant (P less than 0.001). We found no evidence that radiation-induced aberrations varied by age at exposure. These data show that exposure of children or young adults to partial-body fractionated radiation can result in detectable increased frequencies of stable chromosome aberrations in circulating lymphocytes 30 years later, and that these aberrations appear to be informative as biological markers of population exposure.

On the use of laboratory markers as surrogates for clinical endpoints in the evaluation of treatment for HIV infection.

There are strong ethical and practical reasons for hastening decision-making about the efficacy of new treatments for human immunodeficiency virus (HIV) infection. One strategy is to use early markers of disease progression, such as CD4+ lymphocyte levels, as surrogates for ultimate clinical endpoints, such as the development of acquired immune deficiency syndrome (AIDS) or death, in the evaluation of new therapies. We used a simple model of transitions among three health states (well; alive but with an adverse marker; and having experienced a definitive clinical endpoint) to examine the extent to which treatment comparisons based on the surrogate endpoint predict ultimate clinical benefits. With parameters chosen to model the treatment of HIV infection, computer simulations of clinical trials demonstrated substantial time savings by use of the surrogate endpoint. However, reliance on the surrogate led to serious overestimates of ultimate clinical benefit if treatment entailed delayed toxicity or had only transient beneficial effects. Likewise, reliance on the surrogate led to serious underestimates of ultimate clinical benefit when the treatment had no effect on the transition from well to the marker state but did reduce the rates of transition from the marker state to the ultimate clinical endpoint and directly from the well state to the ultimate clinical endpoint.

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix.

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.

Assessment of interaction among three carcinogens on rat mammary carcinogenesis in a factorially designed experiment.

Female Sprague-Dawley rats were given by stomach tube 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6] on the 77th day of age at the rate of 1.6 mg/100 g body weight, or procarbazine [(PCZ) CAS: 671-16-9] on the 84th day of age at the rate of 10 mg/100 g body weight, or 0.5 Gy of total-body x-rays on the 91st day of age, singly or in all possible combinations or no treatment. All rats were studied for mammary carcinogenesis for 370 days after the 84th day of age. Three measures of mammary carcinogenesis were studied. These were the incidence of rats with mammary adenocarcinomas, or mammary fibroadenomas, or mammary neoplasia of either type. Each of these measures was studied also for rats with 2 or more or 3 or more mammary neoplasms. Assessment of possible interaction among the three carcinogens with regard to the incidence of neoplasms was done by time-independent or time-dependent methods, both of which gave remarkably consistent results. For rats with 1 or more adenocarcinomas, 1 or more fibroadenomas, or 1 or more adenocarcinomas and/or fibroadenomas, both methods showed no interaction among the carcinogens, which can, therefore, be considered to have produced additive effects. An exception to this finding of additivity was an apparent synergistic interaction between DMBA and PCZ when the measures of rats with 2 or more, or 3 or more mammary neoplasms of either type, or 3 or more fibroadenomas were analyzed; these analyses, however, were based on relatively small numbers of rats with multiple tumors. Since no interactions were found for the usual measure of carcinogenesis, namely, incidence of rats with 1 or more neoplasms, the overall conclusion is that DMBA, PCZ, and x-ray act additively in the induction of mammary neoplasms in the female Sprague-Dawley rat.

Cancer mortality and radioactive fallout in southwestern Utah.

Cancer mortality was compared between a three-county region in southwestern Utah and the remainder of Utah in an investigation of reported excess cancer risks associated with residence in southwestern Utah during the period of above-ground nuclear tests at the Nevada Test Site. Because most of the fallout in southwestern Utah was deposited during 1953-1957, comparisons were limited to persons born before 1958, and deaths from leukemia and bone cancer during 1955-1980 and from other cancers during 1964-1980. There was no excess risk of cancer mortality in southwestern Utah, for single or grouped sites, with the single exception of leukemia which showed statistically significant odds ratios of 1.45 based on 62 deaths at all ages, and 2.84 based on nine deaths at ages 0-14. The finding for childhood leukemia was based on different time periods and geographic comparisons from those of two earlier studies in which no such excess was found. Mortality from all cancer sites combined was significantly lower in southwestern Utah than in the remainder of the state, even after adjustment for the higher proportion of (lower risk) Mormons in southwestern Utah. The present results, including the positive association for leukemia, are inconsistent with the high excess risks reported by Johnson (JAMA 1984;251:230-6) based on an interview survey of cancer incidence among long-term Mormon residents of southwestern Utah.

White blood cell count and haematocrit as predictors of coronary recurrence after myocardial infarction.

Baseline white blood cell count (WCC) and haematocrit were examined in relation to recurrent coronary events and to all-cause mortality in 2026 persons enrolled in the first Persantin-Aspirin Reinfarction Study (PARIS-1) 2-60 months after myocardial infarction. WCC was strongly related to coronary recurrence (relative risk 3.5 for men with WCC greater than or equal to 9 X 10(9)/l vs men with WCC less than 5 X 10(9)/l) and total mortality (relative risk 2.6). No such relationships were found for haematocrit. WCC correlated also with cigarette-smoking, diuretic use, serum cholesterol and uric acid; however, the associations with coronary recurrence and total mortality persisted on multiple linear and logistic regression analysis including these variables and treatment group (p less than 0.001). WCC is therefore an easily-measured prognostic variable in survivors of myocardial infarction. Furthermore, we suggest that white blood cells may promote myocardial ischaemia by capillary plugging and/or release of toxic oxygen metabolites.

Assessment of interaction between carcinogens in long-term factorially designed animal experiments.

Two substances interact if the combined action results in an effect which is greater than, or less than, that of the sum of the effects produced separately by the substances. Finney (1971, Probit Analysis, 3rd ed. Cambridge: Cambridge University Press) proposed a probabilistic model for simple independent action of two substances for the case of dichotomous outcomes. Using Finney's underlying model, we examine tests for interaction between carcinogens or toxic substances for data from long-term 2 x 2 and 2 x 2 x 2 factorially designed animal experiments. The outcome of interest is the occurrence or not of a certain event (for example, the appearance of a tumor) and the time at which the event occurred. The proposed tests are based on the proportional hazards model. We discuss some simple methods for checking the appropriateness of the proportional hazards assumption. Applications of the tests are made to data from a 2 x 2 and a 2 x 2 x 2 experiment.

Endometrial cancer following treatment for breast cancer: a case-control study in Denmark.

To evaluate the risk of endometrial cancer subsequent to breast cancer, a case-control study was carried out in Denmark. Between 1943-1977, 115 cases of histologically confirmed endometrial carcinoma developed more than 3 months after the diagnosis of a primary breast cancer in 51,638 women. A total of 235 breast cancer patients with no second primary cancer were matched to the cases on age, calendar year of diagnosis, and survival with an intact uterus. Identification of cases and controls relied upon records available in the Danish Cancer Registry. Information on risk factors and reproductive histories was abstracted from hospital records. Increased relative risks (RR) for endometrial cancer were associated with menopausal oestrogen use (RR = 4.9), nulliparity (RR = 2.1), late age at natural menopause (RR = 2.9), and pelvic irradiation (RR = 1.4). No association was apparent for drugs used to treat breast cancer. This study indicates that breast and endometrial cancer share several common aetiologic factors and that studies of second primary cancers have the potential to provide information on risk factors other than those associated with therapy.

Childhood leukemia and fallout from the Nevada nuclear tests.

Cancer mortality data from the National Center for Health Statistics, covering the period 1950 through 1978, were used to test a reported association between childhood leukemia and exposure to radioactive fallout from nuclear weapons tests in Nevada between 1951 and 1958. No pattern of temporal and geographic variation in risk supportive of the reported association was found. Comparison of these results with those presented in support of an association of risk with fallout suggests that the purported association merely reflects an anomalously low leukemia rate in southern Utah during the period 1944 to 1949.