Association of vitamin B12, methylmalonic acid, and functional parameters.

INTRODUCTION: Diagnosis of vitamin B12 deficiency is difficult, as there is no conclusive single test for this disorder. We evaluated the association of serum B12 and methylmalonic acid (MMA) with haematologic parameters and physical and cognitive functioning in an effort to use such clinical parameters to improve the interpretation of serum values. METHODS: We used data of participants > 19 years of age from NHANES 2011-2012 and 2013-2014, a cross-sectional survey in the United States. Functional status was assessed with questionnaires on current health condition, disability, hospital utilisation, cognitive functioning, mental health and depression, and physical functioning. Muscle strength assessed with a handgrip dynamometer was used as a performance parameter. Results were evaluated both for the entire population and participants of Western European descent. Because renal function influences MMA concentrations and is a proxy for both frailty and comorbidity, all results were additionally stratified for individuals with normal vs impaired renal function (eGFR < 60 ml/min). RESULTS: In total, data of 9645 participants (mean age 49 (SD 17) years, 49.3% males) were included. Out of all participants with serum B12 < 140, 140-300, and 301-1000 pmol/l, 56.2%, 13.5%, and 4.1%, respectively had elevated MMA. MMA concentrations were more strongly associated with poor functional status and physical performance than serum B12. We identified a significant and independent association of MMA concentrations, as well as haemoglobin and co-morbidity with muscle strength. CONCLUSIONS/INTERPRETATIONS: A large proportion of individuals with a decreased serum B12 concentration still has normal MMA concentrations. Elevated MMA concentrations were more strongly associated with poor functional performance than serum B12.

Guidelines on the benefit-risk assessment of the presence of phthalates in certain medical devices covering phthalates which are carcinogenic, mutagenic, toxic to reproduction (CMR) or have endocrine-disrupting (ED) properties.

By the new Medical Device Regulation (MDR, EU 2017/745) the use of certain phthalates which are carcinogenic, mutagenic, toxic to reproduction (CMR) or have endocrine-disrupting (ED) properties, above 0.1% by weight (w/w) is only allowed after a proper justification. The SCHEER provide Guidelines on the benefit-risk assessment (BRA) of the presence of such phthalates in certain medical devices. The Guidelines describe the methodology on how to perform a BRA for the justification of the presence of CMR/ED phthalates in medical devices and/or or parts or materials used therein at percentages above 0.1% w/w. They also describe the evaluation of possible alternatives for these phthalates used in medical devices, including alternative materials, designs or medical treatments. Relevant stakeholders e.g. manufacturers, notified bodies and regulatory bodies, can use the guidelines. The approach of these guidelines may also be used for a BRA of other CMR/ED substances present in medical devices. SCHEER noticed that a number of BRA methodologies are theoretically available. However, there is a considerable lack of data needed for the BRA for potential relevant alternatives to be used in medical devices. Therefore, SCHEER encourages manufacturers to generate data of high quality on such alternatives for CMR/ED phthalates in medical devices.

Ion chromatography for the precise analysis of chloride and sodium in sweat for the diagnosis of cystic fibrosis.

BACKGROUND: Measurement of chloride in sweat is an essential part of the diagnostic algorithm for cystic fibrosis. The lack in sensitivity and reproducibility of current methods led us to develop an ion chromatography/high-performance liquid chromatography (IC/HPLC) method, suitable for the analysis of both chloride and sodium in small volumes of sweat. METHODS: Precision, linearity and limit of detection of an in-house developed IC/HPLC method were established. Method comparison between the newly developed IC/HPLC method and the traditional Chlorocounter was performed, and trueness was determined using Passing Bablok method comparison with external quality assurance material (Royal College of Pathologists of Australasia). RESULTS: Precision and linearity fulfill criteria as established by UK guidelines are comparable with inductively coupled plasma-mass spectrometry methods. Passing Bablok analysis demonstrated excellent correlation between IC/HPLC measurements and external quality assessment target values, for both chloride and sodium. With a limit of quantitation of 0.95 mmol/L, our method is suitable for the analysis of small amounts of sweat and can thus be used in combination with the Macroduct collection system. CONCLUSIONS: Although a chromatographic application results in a somewhat more expensive test compared to a Chlorocounter test, more accurate measurements are achieved. In addition, simultaneous measurements of sodium concentrations will result in better detection of false positives, less test repeating and thus faster and more accurate and effective diagnosis. The described IC/HPLC method, therefore, provides a precise, relatively cheap and easy-to-handle application for the analysis of both chloride and sodium in sweat.

Plasma levels of free metanephrines and 3-methoxytyramine indicate a higher number of biochemically active HNPGL than 24-h urinary excretion rates of catecholamines and metabolites.

CONTEXT: A substantial number of patients with head and neck paragangliomas (HNPGLs) have biochemically active tumors, evidenced by increased urinary excretion of catecholamines and metabolites, including 3-methoxytyramine (3MT). It is unclear whether plasma levels of these parameters are more sensitive to detect biochemical activity in HNPGL patients than urinary excretion rates. OBJECTIVE: To compare plasma free levels vs urinary excretion rates of deconjugated 3MT and combined metanephrines (MNs) in patients with HNPGL. PATIENTS AND METHODS: We included 124 consecutive patients with HNPGL for screening of catecholamine excess by measurement of 24-h urinary excretion rates of deconjugated (nor)metanephrine, (nor)epinephrine, dopamine, vanillylmandelic acid, 3MT, and plasma free levels of (nor)metanephrine and 3MT. RESULTS: Plasma free 3MT levels were increased in 35 of the 124 patients (28%), whereas 24-h urinary excretion of deconjugated 3MT was increased in 30 patients (24%) (P=0.13). Plasma free MN levels were increased in seven patients (6%) and urinary deconjugated MN levels in six patients (5%) (P=1.00). Plasma free normetanephrine (NMN) levels were increased in seven patients (6%), and five patients had increased urinary excretion of deconjugated NMN (4%) (P=0.69). Plasma free combined MN levels (NMN, MN, and 3MT) were increased in 41 patients (33%), whereas 24-h urinary excretion rates of deconjugated combined MNs were increased in 33 patients (27%, P<0.05). CONCLUSIONS: The combined levels of free MNs and free 3MT in plasma indicate a higher number of biochemically active HNPGLs than the 24-h urinary excretion rates of these markers.

The kinetics of the tissue distribution of silver nanoparticles of different sizes.

Blood kinetics and tissue distribution of 20, 80 and 110 nm silver nanoparticles were investigated in rats up to 16 days after intravenous administration once daily for 5 consecutive days. Following both single and repeated injection, silver nanoparticles disappeared rapidly from the blood and distributed to all organs evaluated (liver, lungs, spleen, brain, heart, kidneys and testes) regardless of size. The 20 nm particles distributed mainly to liver, followed by kidneys and spleen, whereas the larger particles distributed mainly to spleen followed by liver and lung. In the other organs evaluated, no major differences between the sizes were observed. Size-dependent tissue distribution suggests size-dependent toxicity and health risks. Repeated administration resulted in accumulation in liver, lung and spleen, indicating that these organs may be potential target organs for toxicity after repeated exposure. A physiologically based pharmacokinetic (PBPK) model for nanoparticles which describes the kinetics of silver nanoparticles was developed. Model parameter values were estimated by fitting to data. No clear relation between parameter values and corresponding particle diameters became apparent.

In vitro and in vivo (cyto)toxicity assays using PVC and LDPE as model materials.

The choice for a biomaterial is partly based on the outcome of (cyto)toxicity assays. The rationales behind the selection of certain parameters, such as cell lines, controls, and animals, were evaluated using a positive and a negative control, and one experimental sample designed to induce intermediate toxicity. Extraction and direct contact assays were performed using human epidermal keratinocytes and mouse fibroblasts and mouse epithelial cells. Cell survival was measured with the tetrazolium salt (MTT) reduction assay. In addition, local implantation studies were performed in mice and rats. The positive control induced a high degree of toxicity in all in vitro tests performed, indicating that the toxicity observed in the direct contact assay was due to in situ extraction of toxic components. In the direct contact assay the negative control tested on the mouse fibroblasts resulted in a significant reduction of cell survival. No decrease in cell survival was found using the experimental sample. Subcutaneous implantation studies in mice showed that the positive control material induced a severe degeneration in mice. However, in rats just minimal alterations were noted. The experimental material induced moderate responses only in mice. Our results indicate that the direct contact assay provides limited additional information on the cytotoxicity of materials if certain limitations are not taken into account. For the in vivo implantation assay mice were superior to rats in detecting local toxic responses.

Study to determine the presence of antipolymer antibodies in a group of Dutch women with a silicone breast implant.

OBJECTIVE: To determine whether there exists a population of Dutch women with a high prevalence of antipolymer antibodies (APA) and severe health complaints/symptoms, and exposure to a silicone breast implant (SBI). As the antigen-specific nature of the antipolymer antibody has not yet been established, we refer to the term polymer binding immunoglobulins. METHODS: The study population was selectedfrom a voluntary registry of SBI recipients of a Dutch consumers organisation. The final selection was based on the severity of self-reported complaints in a questionnaire. A total of 42 SBI recipients were included in the study, clinically examined and blood samples were obtained. RESULTS: In 12 of 42 SBI recipients an increase in the level of polymer binding immunoglobulins was detected compared to a negative reference sample, 3 of these 12 showing a positive and 9 a weakly positive response. In 3 out of 12 non-SBI recipients, included for control on the performance of the APA assay, an increased level of polymer binding immunoglobulins was demonstrated, 2 of these 3 showing a positive and 1 a weakly positive response. The study population of SBI recipients was categorised in severity subgroups (limited, mild, moderate, advanced) based on the fuctional capacity and the physicians general assessment of pain and disease activity. Most (34 of 42) SBI recipients belonged to the limited severity subgroup. CONCLUSION: Our methods failed to select a group of severely symptomatic Dutch SBI recipients reported to have a high prevalence of polymer binding antibodies. A discrepancy was present between the self reported severe complaints and the observed mild clinical symptoms. In the group of SBI recipients with self reported severe complaints recruited we did not find a high prevalence of polymer binding immunoglobulins. SBI exposure (mean 17 years) did not result in induction of polymer binding immunoglobulins in this minimal symptomatic study group.

Medical devices manufactured from latex: European regulatory initiatives.

In Europe the marketing of medical devices manufactured from latex is regulated by directives describing the essential (safety) requirements that products have to fulfill to obtain marketing approval. This paper describes the general requirements for marketing medical devices in Europe and, more specifically, the requirements for products manufactured from natural rubber latex. The requirements for marketing medical devices can be fulfilled by using the relevant harmonized European standards. These standards are regularly under revision to incorporate the latest scientific developments. For certain devices, for example, latex medical (examination and surgical) gloves, specific standards have been published. Medical devices manufactured from latex pose a serious problem because of the risk of induction of allergy both against the latex proteins inherently present (type I or immediate type allergy) and against chemicals added during processing (type IV or delayed type hypersensitivity) present as residues in the latex products. So, besides requirements for product quality in terms of barrier properties, strength, and sterility, the main focus consists of the allergy-inducing properties of the latex products. Recent developments have reopened the discussion on the value of total protein versus allergen determination in latex medical gloves. However, as long as minimal levels needed for both sensitization and elicitation have not been established, a safe maximum level for leachable proteins/allergens in latex products cannot be determined. A European Commission guidance document on the latex allergy problem is currently being drafted by experts from Competent Authorities.

Developmental toxicity but no immunotoxicity in the rat after prenatal exposure to diethylstilbestrol.

Studies with dioxins and PCB's have shown that the developing immune system may be especially vulnerable to xenobiotics during the perinatal period. However, current guidelines for reproductive toxicity testing do not include immune parameters. In the present study, we have explored the usefulness of including immune parameters within the prenatal developmental toxicity study in rats, using the treatment protocol as described in the OECD 414 developmental toxicity test guideline. In addition, the experimental protocol was enhanced by including ten dose groups to facilitate dose-response analysis. Diethylstilbestrol (DES) was used as the model compound, as it is known to be toxic both for embryofetal development and for the immune system. The results show developmental toxicity in terms of decreased fetal survival and decreased pup body weight in the presence of reduced maternal food consumption and reduced body weight gain. However, immune parameters, including histopathology, hematology, and antibody responses to sheep red blood cells (SRBC) in pups at 4 weeks of age were uncompromised. It is speculated that rather than the prenatal exposure protocol used here, the generation study design with both pre- and postnatal exposure may be preferable as a general screen to detect developmental immunotoxic injury after xenobiotic exposure.

Comparison of dose-responses of contact allergens using the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has been used as a method for the prediction of skin sensitizing potential for over 30 years. Besides hazard identification, risk assessment of sensitizing chemicals requires the assessment of potency. For the determination of potency based on lowest effective dose levels, dose-response studies are required. In the standard GPMT a single concentration is used for intracutaneous and topical induction and the assay provides a qualitative assessment of allergenicity. This paper presents data derived from quantitative evaluation of the sensitizing potency of chemicals in the GPMT, based on multiple concentrations. We performed the GPMT in accordance with the original procedure of Magnusson and Kligman; and included in this procedure a range of intradermal and topical concentrations for induction. Three allergens with different sensitizing potencies, diethylamine (DEA), tetramethyl thiuram disulfide (TMTD) and zinc dimethyl dithiocarbamate (ZDMC) were tested. The data obtained with this test procedure were compared to data we previously obtained using the local lymph node assay (LLNA). Both the GPMT and the LLNA showed dose response relationships for the three chemicals tested. For the chemicals tested, both tests differed in the relative potencies based on benchmark concentrations. While both tests ranked DEA as the least potent allergen, the GPMT ranked ZDMC more potent than TMTD, the reverse being found in the LLNA. The nature of the data provided in the LLNA makes it likely that benchmarks as defined with this test are more reliable than that defined in the GPMT. However, further validation with human data is necessary.

Long-term cannulation of the vena cava of rats for blood sampling: local and systemic effects observed by histopathology after six weeks of cannulation.

A cannulation system with fixation by a metal cuff around the tail was used for blood sampling. The cannula was guided subcutaneously and positioned in the vena cava after entering the body via the femoral vein. Histopathology was performed after long-term cannulation of up to 35 and 45 days. The presence of the cannula in the vena cava induced endothelial hypertrophy and hyperplasia accompanied by stromal hypertrophy. The endothelial activation was not limited to the vena cava but was also observed in both the cannulated vena iliaca and the contralateral control vena iliaca, the latter showing only minor alterations. In the lung, thrombi were noted in the larger lung arteries; and foreign body emboli, probably situated in the alveolar septi, could be detected occasionally. Inflammatory reactions in the tail at the site of cuff fixation consisted of a mixture of acute and chronic inflammatory responses. The chronic inflammation extended into the tail muscles, as shown by the presence of fibrous tissue associated with muscle degeneration. In conclusion, prolonged venous cannulation in rats resulted in local alterations in the veins, small emboli in the lungs and a moderate to marked inflammation in the tail. However, the procedure itself was well tolerated by the animals.

Retrovirus type C in the mouse bladder carcinoma cell line MBT-2.

PURPOSE: The presence of replicating type C retrovirus in MBT-2 mouse bladder carcinoma cells is reported. This MBT-2 tumor cell line is nowadays globally distributed. The cells have been and are still used to study various aspects of bladder cancer. While studying the phagocytic capacity of MBT-2 cells for BCG organisms by electron microscopic methods, the presence of this retrovirus was noticed. MATERIALS AND METHODS: MBT-2 cells that were cultured in vitro as well as cells from intravesically and intradermally grown MBT-2 tumors from syngeneic mice were investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques. RESULTS: All samples including the earliest generation MBT-2 cells that could be traced from stocks of other research groups contained the C type retrovirus, suggesting a contamination in all available generations of the MBT-2 cell line. CONCLUSIONS: As this tumor cell line is widely used in immunologic studies of the response to bladder cancer, it is important to consider the possible presence of type C viruses and associated antigens, since they could contribute to or interfere with the responses being measured. Studies should be initiated to determine whether viral antigen expression is involved in the immune rejection of MBT-2 bladder cancer. As a consequence, clinical implementation of immunological treatment strategies should not be based on results obtained with the MBT-2 model alone, but preferably should be confirmed with other (bladder) carcinoma models.

A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of the uncertainty margins.

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-gamma and interleukin-4.

The local lymph node assay (LLNA) is a new and promising test in mice used to identify contact allergens by means of dermal exposure. Experimentally this assay, which comprises a sensitizing phase only, is also used to identify respiratory allergens. Another, experimentally used test in mice to identify allergens is also based on dermal exposure, but comprises both a sensitizing and effector phase. In this latter test, it has been shown that contact allergens preferentially induce a T-helper 1 (TH1) response, whereas respiratory allergens preferentially induce a T-helper 2 (TH2) response. These responses can be discriminated on the basis of cytokine production, such as IFN-gamma, which is produced by TH1 cells, and IL-4, which is produced by TH2 cells. The aim of the study was to establish whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen. To this end, LLNA responses to the contact allergen dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) were determined using IFN-gamma and IL-4 mRNA expression and production as parameters. Topical application of TMA resulted in a threefold higher lymphocyte proliferation compared to DNCB 3 and 5 days after the first application, while a similar proliferation was found from Day 7 and onward. RT-PCR showed a similar induction of IFN-gamma and IL-4 mRNA expression. While both DNCB and TMA induced IFN-gamma production, TMA but not DNCB induced IL-4 production. Thus, only IL-4 production seemed a suitable parameter to discriminate between the two compounds. In a second study, the respiratory allergens toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA) were also assayed 7 days after the first application. Topical application of DNCB and PA resulted in a similar lymphocyte proliferation, while application of TMA and TDI resulted in a 1.8-fold higher proliferation. IFN-gamma production was similar for DNCB, TMA, and TDI, and fourfold lower for PA, while IL-4 production was similar for TMA, TDI, and PA, and 24-fold lower for DNCB. In summary, both studies showed induction of IL-4 production by respiratory allergens, with little or no induction by the contact allergen, holding promise for the possibility of identifying respiratory allergens within the LLNA by measuring IL-4 production 7 days after the first application.

Detection of immunotoxicity of benzo[a]pyrene in a subacute toxicity study after oral exposure in rats.

In an extended OECD 407 study protocol, including immune parameters, male Riv:Tox Wistar SPF rats were treated for 35 days with benzo[a]pyrene (B[a]p) (3, 10, 30, or 90 mg/kg body weight) by gavage. Oral administration of B[a]p in rats resulted not only in general toxicity, as indicated by the effects on body weight, but also in immunotoxicity, as indicated by the effects on bone marrow, thymus, spleen, and lymph nodes. Oral B[a]p induced a dose-related decrease in thymus weight (at 10, 30, and 90-mg/kg). Lymph node weights (popliteal, mandibular, and mesenteric) were decreased in the 90-mg/kg rats only. Histologically, indications for cortical atrophy were noted in the thymuses of the 30- and 90-mg/kg dose groups, which was confirmed by morphometric analysis. Nucleated spleen and bone marrow cell counts were decreased in the 90-mg/kg group. Both the absolute number (90 mg/kg) and relative number (10, 30, and 90 mg/kg) of B cells in the spleen were decreased. Red blood cell (RBC) and white blood cell (WBC) counts were significantly decreased; for the WBC at 90 mg/kg, and for the RBC at 10, 30, and 90 mg/kg. The absolute number of lymphocytes and eosinophilic granulocytes was decreased in the 90-mg/kg group, while the absolute number of monocytes was increased in the 10- and 30-mg/kg dose groups. Serum immunoglobulin levels showed a decrease of IgM and IgA after treatment of the animals with 30 and 90 mg/kg, respectively. The highest dose of B[a]p treatment (90 mg/kg) resulted in a significant decrease of natural killer (NK)-cell activity in the spleen. Most toxic effects were only observed in the highest-dose group (90 mg/kg), but compared to the general toxicity, some parameters indicating immunotoxic effects were also affected at lower doses (10 and 30 mg/kg). In conclusion, immunotoxicity of B[a]p can be detected using parameters of the immune system such as described in the recently updated OECD 407 guideline. In the present study thymus weight changed and spleen B-cell populations were affected at a dose of 10 mg/kg, a level where no overt general toxicity was noted.

Effects of salmeterol on host resistance to Trichinella spiralis in rats.

Salmeterol is a long-acting beta2-adrenoreceptor agonist. The compound has previously been screened for immunotoxic potential in a repeated dose toxicity study in rats for 28 days. The total serum IgG levels were increased at dose levels of 2 and 10 mg/kg/day. Presently, salmeterol was studied in an immune function assay addressing the host resistance to Trichinella spiralis parasites. Rats were daily treated with salmeterol for 28 days at dose levels of 0, 2, 6 and 10 mg/kg/day. On day 29, the animals were infected with T. spiralis parasites. After six weeks, host resistance was examined. The numbers of T. spiralis muscle larvae in the tongue nor the inflammatory reactions around the encapsulated larvae were affected by salmeterol treatment. The yield of muscle larvae in the whole carcass was not changed either. The IgM, IgA and IgE antibody responses to T. spiralis were unaffected. Only at the highest dose level tested, the anti-T. spiralis IgG antibody response was decreased significantly. However, salmeterol's interference with the generation of anti-T. spiralis antibodies of the IgG subclass apparently did not adversely affect the resistance to infection.

Risk assessment and immunotoxicology.

In general toxicity testing, maximal acceptable concentrations are derived from no-observed adverse effect levels (NOAEL) in rodents. Risk assessment then considers safety factors for the interspecies difference, and intraspecies variability. This approach can be used for assessing maximal acceptable concentrations for chemicals inducing direct immunotoxicity, resulting in e.g. reduced resistance to infections. As for predictive testing of chemicals in terms of sensitization, laboratory animal data are mostly used for risk assessment as well. Generally, the assessment of risk for chemicals that induce contact sensitivity is limited to hazard identification, and risk management is restricted to labeling. An alternative type of evaluation of the risk of adverse effects due to exposure to immunotoxic chemicals may be the so called parallellogram approach. In this parallellogram there are four cornerstones, one of which is the health effect of exposure to a chemical, assessed as an endpoint (e.g. infection model) in experimental animals, and another the quantitative prediction of this endpoint in humans. The other cornerstones are assays of parameters that are relevant to the mechanism of the adverse effect in experimental animals and humans, and are used for species comparison. Species comparisons between the animal species used for hazard identification and humans are crucial for extrapolation of animal data to the human situation. This approach can be used to provide relevant information on the dose-response relationship in humans. In concert with information on actual exposure, such data can then be used for the characterization of risk for adverse health effects in humans. Such approaches have been used for chemicals that exert direct immunotoxic activity (bis(tri-n-butyltin)oxide (TBTO)), and may hold promise for the risk evaluation of chemicals that exert skin sensitizing properties.

Response of sensitive and resistant IgM immunocytomas to cis-diamminedichloroplatinum (II) does not correlate with the platination level or with the formation or removal of DNA adducts.

An IgM immunocytoma cell line sensitive to cis-diamminedichloroplatinum(II) (CDDP) and a subline with acquired resistance were grown in LOU/M rats. In a previous study with such rats that had been treated with a high dose of CDDP (10 mg/kg) the tumors did not show differences in cellular platinum content or DNA-adduct levels, either immediately after treatment or 24 h later. Recently, this high dose was found to overcome resistance. Therefore, the study was repeated with a 10-fold lower dose (1 mg/kg, i.v.). At 1 and 24 h after treatment, tumor and kidney tissue were assayed for cellular platinum (atomic absorption spectroscopy, AAS) and DNA platination (immunochemical detection of the four CDDP-DNA adducts). The results were compared with previous data. All tissues showed a linear response to dose with regard to platinum uptake as well as adduct formation, with no quantitative difference being seen between the tumors. Also the relative occurrence of the four adducts was very similar. Between 1 and 24 h, in tumors a substantial decrease occurred in both platinum content and adduct level; the kidneys showed little reduction, if any. At the lower CDDP dose a somewhat larger loss of platinum and removal of DNA adducts was observed for the resistant tumor, but these differences could be explained by "dilution", as this tumor continues to grow after low-dose treatment (about 20% within 24 h). Since the strong difference observed between the tumors in sensitivity to CDDP cannot be attributed to differences in CDDP uptake, efficiency of adduct formation, or repair capability, other mechanisms are held responsible.

An immunotoxicity screening study on salmeterol in rats.

Salmeterol, a long-acting beta 2-adrenoreceptor agonist without known immunotoxicity, was studied in a 28-day repeated dose toxicity test in Wistar rats. Several immunotoxicity screening parameters were incorporated in the study protocol to investigate the immunotoxic potential of the compound. Male rats were orally treated with 0, 0.2, 2 and 20 mg salmeterol/kg body weight/day. At the 20 mg/kg/day dose level, intubation errors occurred because the animals tried to resist intubation. Some of these animals died intercurrently. Therefore, the magnitude of the dose was lowered to 10 mg/kg/day at day 9 of treatment. Body weight and bone marrow cellularity were not affected. Hematological parameters were not altered either, except for platelet counts, that were decreased at all dose levels. Also liver weights were decreased at all dose levels tested. Absolute thymic weights were decreased at the 2 and 20/10 mg/kg/day dose levels. No treatment-related (histo)pathological lesions were seen in the (non)lymphoid organs. Serum IgM levels were increased at the 0.2, and IgG at the 2 and 20/10 mg/kg/day dose levels, respectively. B cell numbers in the spleen were decreased at all dose levels tested. The data indicate that the test battery applied to salmeterol is able to detect low immunotoxic potential. Further research is needed to elucidate whether salmeterol interferes with immune responses in rats upon antigenic challenge.

Design of immunoliposomes directed against human ovarian carcinoma.

Factors (protein/lipid ratio, pH of incubation medium, incubation time, anchor molecule density in the bilayer) affecting the covalent binding of anti-ovarian carcinoma Fab' to liposomes containing the anchor molecule MPB-PE (N-(4-(p-maleimidophenyl)butyryl)phosphatidylethanolamine) were explored. Standard experimental conditions were chosen and information on the relevant physicochemical parameters of the liposome dispersions was collected (mean particle diameter, size distribution, charge). The reproducibility of standard immunoliposomes prepared in subsequent batches in terms of Fab' binding, particle size and charge was established. In addition, preservation of immunoreactivity, no marker loss, and no aggregation/fusion was found for the standard immunoliposomes over a period of at least 3 weeks at 4 degrees C. In vitro up to 35,000 immunoliposomes were estimated to bind per human ovarian carcinoma cell. Internalization of the immunoliposomes could not be demonstrated. Electron micrographs showed binding of specific immunoliposomes to human ovarian carcinoma cells growing intraperitoneally in athymic nude mice.