Modeling anti-KLH ELISA data using two-stage and mixed effects models in support of immunotoxicological studies

During preclinical drug development, the immune system is specifically evaluated after prolonged treatment with drug candidates, because the immune system may be an important target system. The response of antibodies against a T-cell-dependent antigen is recommenced by the FDA and EMEA for the evaluation of immunosuppression/enhancement. For that reason, we developed a semiquantitative enzyme-linked immunosorbent assay to measure antibodies against keyhole limpet hemocyanin. To our knowledge, the analysis of this kind of data is at this moment not yet fully explored. In this article, we describe two approaches for modeling immunotoxic data using nonlinear models. The first is a two-stage model in which we fit an individual nonlinear model for each animal in the first stage, and the second stage consists of testing possible treatment effects using the individual maximum likelihood estimates obtained in the first stage. In the second approach, the inference about treatment effects is based on a nonlinear mixed model, which accounts for heterogeneity between animals. In both approaches, we use a three-parameter logistic model for the mean structure.     

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks

OBJECTIVE: The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. METHODS: In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. RESULTS: No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. CONCLUSION: The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.     

Relationship between the energy status of Daphnia magna and its sensitivity to environmental stress

This work tested the hypothesis that animals with a high energy status are more successful in dealing with stress than animals with a low energy status. Daphnids (Daphnia magna) were reared for 2 weeks in four different concentrations of food. Survival was not affected by food supply, and growth and reproduction increased with increasing food ration. This increase correlated well with the energy status, as was measured by scope for growth on day 15. After 2 weeks, the daphnids in the four different food ration groups were exposed for another 2 weeks to a range of increased salinities or cadmium concentrations, while remaining in their respective food concentrations. In the salinity groups, survival, growth, or reproduction were not influenced at low salinities. Exposure to higher salinity significantly decreased survival and reproduction, but this decrease was more pronounced in the highest food concentrations. In the cadmium exposed daphnids, cadmium content increased with increasing exposure concentrations, but accumulation was independent of food rations. Cadmium exposure significantly decreased survival, growth, and reproduction and this decrease again was more pronounced with increasing food concentration. Thus, the high energy status of the daphnids from the high food concentrations at the start of the exposure did not provide an increased capacity to cope with additional stress. Instead, the sensitivity of the daphnids to stress increased with increasing food ration. This increased sensitivity is likely to be the result of a change in life history from emphasizing survival at low food supply to stressing reproduction at high food supply.     

Dermal exposure to cyclophosphamide in hospitals during preparation, nursing and cleaning activities

Objectives: To determine levels of potential and actual dermal exposure to cyclophosphamide (CP) during performance of oncology-related tasks in hospitals and to investigate the relationship with potential sources and surface contamination levels of CP. Methods: Dermal exposure to CP was determined for tasks with potential exposure to CP: preparation of CP, decanting of patients urine, washing of the patient, removal of bed sheets of treated patients and cleaning of patients toilets on oncology wards. Exposure was assessed by the collection of nitrile and latex protective medical gloves (potential exposure), washing of hands (actual exposure), from cotton pads attached to (un)covered forearms (potential or actual exposure) and a wipe sample of the forehead (actual exposure). Bulk samples (i.e. application fluids and patients excreta) and possible contact surfaces were monitored to assess the amount of CP available for dermal exposure. Results: Pharmacy technicians, oncology nurses and cleaning personnel showed actual and potential dermal exposure to CP during performance of their daily duties. Exposure occurred predominantly on the hands and sporadically on the forehead and forearms. Although all nurses used gloves during handling of patients urine and sometimes during the other nursing tasks, skin underneath gloves was repeatedly contaminated. Results of tests on bulk and surface contamination samples confirmed that patients intravenously treated with CP excrete the unmetabolised drug, which could subsequently lead to dermal exposure of hospital personnel. A clear relationship was found between dermal exposure levels and direct sources of exposure for all tasks, except for handling patients urine. Conclusions: We demonstrated for the first time that actual dermal exposure to CP is common among oncology nurses working with patients treated with this anti-neoplastic drug. Pharmacy technicians and cleaning personnel, on the other hand, are potentially exposed to CP, and protection provided by gloves seemed to be sufficient.     

New lymphogranuloma venereum Chlamydia trachomatis variant, Amsterdam

We retrospectively conducted a study of men who have sex with men who visited the Amsterdam, the Netherlands, sexually transmitted diseases clinic from January 2002 to December 2003 and had rectal Chlamydia trachomatis infections. We found that symptomatic (73%) as well as asymptomatic (43%) patients were infected with a new C. trachomatis LGV variant.     

Occupational chemical risk: elements for a diagnostic in Spain

BACKGROUND: Lack of information and accurate knowledge about the properties of chemicals and the exposure derived from its use; hinder health and safety measures to protect exposed workers. The aim of this study is to identify the elements that will help us draw a balance of chemical hazards in Spain and register the most dangerous chemicals at the workplace (by sector or field of activity). METHODS: Study of secondary sources, identification of risk perception by workers' representatives and study of the approach on chemical risk in risk assessments. The last step is to set up a Chemical Exposure Database arranged by industrial sectors. RESULTS: Great volumes of production and consumption of chemicals, generalization of exposure, but insufficient data on specific use; little information is provided on specific dangerous factors of commonly used chemicals; chemicals are barely identified in risk assessments. High levels of exposure in industrial sectors; damage is not adequately registered; there are difficulties in risk perception. Set-up of a preliminary database on exposure by sector of activity. CONCLUSIONS: Data is collected; information sources and procedures, which allow a forecast of chemical risk, are identified. A database on exposure by sector of activity is developed (to be checked and enhanced as new evidence is found).     

Similar left and right ventricular sarcomere structure after support with a left ventricular assist device suggests the utility of right ventricular biopsies to monitor left ventricular reverse remodeling

BACKGROUND: To evaluate whether the morphology of the contractile filaments in cardiomyocytes of patients with end-stage heart failure, treated with a left ventricular assist device (LVAD), is identical in the left- and right ventricle (LV, RV) and in the interventricular septum (IVS) and can be monitored by biopsies taken with a bioptome. The application of an LVAD as a bridge to recovery of cardiac function requires monitoring of myocyte recovery. The use of RV biopsies for this purpose might be feasible, if morphologic findings in the RV coincide with those in the LV. METHODS AND RESULTS: At the time of heart transplantation, myocardial biopsies of LV, RV and IVS from 13 patients after LVAD support were compared using immunohistochemistry with monoclonal antibodies against contractile proteins. Additionally, in five of these patients, small biopsies obtained with a diagnostic bioptome were compared with large transmural biopsies of the same region. Hemodynamic monitoring was performed when the patients were fully recovered from the implantation, to rule out persistent RV failure. The staining pattern of actin, myosin, tropomyosin, troponin T and C was identical in the biopsies of LV, RV and IVS. Small biopsies taken with a bioptome appeared to be representative for the larger biopsies. Hemodynamic monitoring showed absence of RV failure in our study group. CONCLUSION: In the absence of RV failure, morphology of the contractile myofilaments after LVAD support for 215+/-143 days is identical in LV, RV and IVS. This may allow monitoring of the possible occurrence of LV reverse remodeling by RV biopsies.     

Medical management for termination of second and third trimester pregnancies: a comparison of strategies

OBJECTIVE: Misoprostol and sulprostone are prostaglandins that can be used for the termination of second and third trimester pregnancy. The aim of the present study was to compare the effectiveness of both agents for the termination of second and third trimester pregnancy in cases of congenital or genetic abnormalities, and for the induction of labour in cases of intra-uterine foetal death. STUDY DESIGN: We collected data from all women who had been treated with misoprostol in the second or third trimester of pregnancy between January 2001 and July 2002 in cases of congenital or genetic abnormalities, and for the induction of labour in cases of intra-uterine foetal death. In cases where the foetus was alive, misoprostol was usually (77%) combined with mifepristone. Women were matched to women who had been treated with sulprostone for termination of second and third trimester pregnancy before 2001. We matched for hospital, previous vaginal delivery, intra-uterine death and duration of pregnancy. The primary outcome measure was time to delivery. RESULTS: Since the treatment effect was different in patients in whom labour was induced for intra-uterine death and patients in whom labour was induced while the foetus was alive, the analysis was stratified for this parameter. In 94 patients with intra-uterine death, there was no significant difference in time to delivery, blood loss, operative removal of the placenta and need for pain relief between misoprostol and sulprostone. In vital pregnancy (n = 96), time to delivery was significantly shorter in the misoprostol group. The relative risk for haemorrhage exceeding 1000 ml in this group was 0.40 (95% confidence interval, CI, 0.13-1.2). We observed no significant differences with respect to operative removal of the placenta or need for pain relief. CONCLUSION: In cases of intra-uterine death, the effectiveness of misoprostol for termination of pregnancy is comparable to that of sulprostone. In vital pregnancy, combination of mifepristone and misoprostol is more effective than sulprostone alone.     

Temporary losartan or captopril in young SHR induces malignant hypertension despite initial normotension

BACKGROUND: Exposure of normotensive rats to angiotensin-converting enzyme (ACE) inhibitors in early life causes hypertrophy of intrarenal arteries. Similar defects have been found in knockout mice lacking angiotensinogen, ACE, or angiotensin II type 1 (AT1) receptors. On the other hand, transient inhibition of the renin-angiotensin system from 2 weeks of age in spontaneously hypertensive rats (SHR), either with ACE inhibitors or with AT1 receptor antagonists partially prevents the increase in blood pressure. However, permanent treatment of SHR from conception onwards with ACE inhibitors completely prevents hypertension. Although these studies demonstrated protection from hypertension-induced changes in the heart and large arteries, renal arteries were not studied and follow-up did not extend beyond 6 months of age. We postulated that while brief exposure to ACE inhibitors or AT1 receptor antagonists in young SHR would temporarily decrease blood pressure, it would also be associated with development of intrarenal arterial malformation, and ultimately have deleterious effects. METHODS: Direct effects on intrarenal arterial morphology of an ACE inhibitor (captopril, 100 mg/kg/day) and an AT1 receptor antagonist (losartan, 50 mg/kg/day), administered from the last week of gestation until 8 weeks of age were examined in SHR. After stopping treatment at 8 weeks, we continued to monitor blood pressure until spontaneous death. RESULTS: Systolic blood pressure at 8 weeks was normalized by captopril and losartan (SHR control 187 +/- 8 mm Hg; captopril 118 +/- 5 mm Hg; and losartan 120 +/- 9 mm Hg). However, by 30 weeks, blood pressure had increased to control SHR levels. At 4 weeks, the media of renal arteries and arterioles was hypertrophied. Marked smooth muscle cell hyperplasia of cortical arteries resulted in significantly increased wall thickness by 8 weeks, despite similar external diameter. Arterial wall structure was disrupted, with fragmentation of elastic fibers and irregular distribution of collagen type I fibers. After stopping treatment, the rats gradually began to show poor health and all had died by 1 year of age, while all 1-year-old control SHR females were in good health. The cause of morbidity and mortality in the rats treated in early life was clearly malignant hypertension. Severe hypertrophy of renal arterioles was found, as well as cerebral hemorrhage. CONCLUSION: Despite initial normalization of blood pressure interference with the renin-angiotensin system during a crucial stage of development in SHR can initiate marked smooth muscle cell hyperplasia and disruption of the wall structure of the intrarenal arteries. Subsequent progression of this intrarenal process after cessation of treatment suggests an independent process that eventually results in malignant hypertension and early death.