Determination of amoxicillin in plasma samples by capillary electrophoresis

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C₁₈ cartridges followed by elution with water–methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 μg l⁻¹ by the FASI technique.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.     

Hydrophobic interaction chromatography and capillary zone electrophoresis to explore the correlation between the isoenzymes of salivary α-amylase and dental caries

High-performance hydrophobic interaction chromatography (HIC) and capillary zone electrophoresis (CZE) were utilized in an effort to find the correlation between the composition of some isoenzymes of human salivary α-amylase (HSA) and dental caries. The mixture of more than three isoenzymes of HSA, fractionated from human parotid saliva with HIC, was further separated by CZE at the optimum pH 6.50. The composition and relative quantity of these isoenzymes were compared between two groups of individuals with different caries-susceptibility. It is found that the present frequency of peak II on CZE in the caries-free group was higher than that in the caries-active group and the relative quantities of peak III and peak IV showed remarkable differences (p<0.05) between the two groups. These results may indicate that the composition of HSA isoenzymes is related to the occurrence of dental caries. However, more work should be done to further affirm this correlation between the isoenzymes of salivary α-amylase and dental caries.     

Analysis of gamma radiation-induced damage to plasmid DNA using dynamic size-sieving capillary electrophoresis

Bacterial plasmids and the chromosomal DNA of many organisms adopt naturally the negatively supercoiled conformation. Therefore, the irradiation of such plasmids could be used to model conformational changes of chromosomal DNA associated with externally-induced damage. We have applied dynamic size-sieving capillary electrophoresis (CE) to monitor the damage of three DNA plasmids, over an unprecedented base pair (bp) size range (2870–27 500 bp), upon exposure to γ-radiation (20–400 Gy). Predominantly, CE with UV absorbance detection in the absence of DNA intercalating dyes was employed to preclude undesirable, induced plasmid conformational changes. Plasmid samples and their enzymatic digestion products were analyzed using both CE and slab gel electrophoresis (SGE) in order to verify the conformation of sample components. Relative to SGE, CE analyses revealed more fine structural features of plasmid degradation.     

Capillary zone electrophoresis for the determination of atovaquone in serum

A rapid and simple capillary zone electrophoresis (CZE) method has been developed for the determination of atovaquone in serum. The drug was extracted from equine serum–chloroform (1:3, v/v) at greater than 80% recovery and assayed in buffer containing 25 mM sodium borate (pH 9.1) and 25% acetonitrile. A 100 μm I.D. fused-silica capillary was used and the detection was by UV-diode array at 254 nm; the migration time was approximately 8 min. Intra- and inter-assay variabilities were less than 7.8% and 5.8%, respectively, and the accuracy of the assay (expressed as % bias) ranged from 4.5 to −5.2%. The working assay range was from 2 to 100 μg/ml. This sensitivity could be increased by concentrating during the extraction procedure. Replacement of acetonitrile with 75 mM surfactant 3-(dimethyldodecylammonio)propanesulfonate gave similar sensitivity and provided an additional option to facilitate the separation of atovaquone on multiple-drug samples.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS–PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107 000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol–diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445–480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M_r 260 000) after silver staining and Coomassie staining of 4–15% gradient SDS–PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS–PAGE bands and the apo B-48 in a protein range of 0–3 μg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.     

Alkaline single-cell gel electrophoresis and human biomonitoring for genotoxicity: a pilot study on breast cancer patients undergoing chemotherapy including cyclophosphamide

Alkaline single-cell gel electrophoresis (the ‘comet assay') was used to evaluate DNA damage in lymphocytes from 17 breast cancer patients before and 1–21 h after chemotherapy including cyclophosphamide (600–1800 mg/m²). In order to control for the experimental variability over time, freshly isolated lymphocytes from female mice given physiological saline or cyclophosphamide (150 mg/kg b.wt.) were included as ‘internal standards' in each individual electrophoresis run. There was an upward tendency of DNA damage in the mouse lymphocytes over the study period, but cyclophosphamide was constantly found to induce significant damage at all time points investigated (1–48 h). Although patients given up to 11 prior cycles of chemotherapy showed the same basal level of DNA damage as the patients coming to the clinic for their first treatment, the chemotherapy given at the time of the present blood sampling was associated with significant DNA damage in most samples. Considerable interindividual variations were observed both before and after the treatment. DNA single-strand breaks and alkali-labile sites in peripheral lymphocytes as evaluated by the comet assay seem to be useful molecular biomarkers for exposure to DNA damaging agents when monitoring ongoing exposures, but less impressive when monitoring accumulated exposures, at least in patients given high doses of cyclophosphamide and other antineoplastic agents.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.     

Upregulation of dihydropyrimidinase-related protein 2, spectrin α II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats—A proteomics approach

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24 h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18–22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3–46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin α II chain (rat fragment, also known as α-fodrin or non-erythroid α chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24 h after the induction of cerebral ischemia.     

Wilm's Tumor-1 Protein Levels in Urinary Exosomes from Diabetic Patients with or without Proteinuria

Background

Podocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilm''s tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline.

Methods

The expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1.

Results

WT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m².

Conclusion

The predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy.     

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2⁺ donors, but no responses were detectable in 9 HLA-A2^– healthy donors or 4 HLA-A2^– melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.     

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Incidence and Risk of Proteinuria with Aflibercept in Cancer Patients: A Meta-Analysis

Background

Aflibercept is a human recombinant fusion protein with antiangiogenic effects that functions as a decoy receptor to bind vascular endothelial growth factor A. Proteinuria is one of its major adverse effects with a substantial variation in the incidence rate, and the overall risk of proteinuria has not been systematically studied. We performed a meta-analysis of published clinical trials to quantify the incidence and relative risk of proteinuria in cancer patients treated with aflibercept.

Methods

The electronic databases were searched, including PubMed, Embase, Cochrane databases, and ASCO (American Society of Clinical Oncology) abstracts. Eligible studies were phase II and III prospective clinical trials of cancer patients treated with aflibercept with toxicity data on proteinuria. Overall incidence rates, relative risk (RR), and 95% confidence intervals (CI) were calculated using fixed or random effects models depending on the heterogeneity of the included studies.

Results

A total of 4,596 patients with a variety of solid tumors from 16 prospective clinical trials were included for the meta-analysis. The overall incidences of all-grade and high-grade proteinuria in cancer patients were 33.9% (95% CI: 27.3–42.1%) and 7.9% (95% CI: 6.1–10.2%). The relative risks of proteinuria of aflibercept compared to control were increased for all-grade (RR = 1.41, 95% CI: 1.13–1.77) and high-grade (RR = 6.18, 95% CI: 3.78–10.12) proteinuria. The risk of developing all-grade and high-grade proteinuria with aflibercept was substantially higher than that of bevacizumab (all-grade: RR 1.85, 95% CI: 1.63–2.11; high-grade: RR 2.37, 95% CI: 1.84–3.05).

Conclusions

Aflibercept is associated with an increased risk of developing proteinuria. Appropriate monitoring and treatment is strongly recommended to prevent potential renal damage. Future studies are still needed to investigate the risk reduction and possible use of aflibercept in cancer patients.     

On-line post-capillary affinity detection of immunoglobulin G for capillary zone electrophoresis

To address the quality issues of antibody manufacturing, post-capillary affinity detection of immunoglobulin G (IgG) is developed for capillary zone electrophoresis. In analogy to a two-dimensional separation system, capillary zone electrophoresis (CZE), as the first dimension, resolves IgG variants based on their differences in molecular structure. IgG variants separated by CZE are discriminated against other serum and cellular proteins by affinity complex formation with protein A binding fragment in a post-capillary reactor. The analytical power of post-capillary affinity detection is demonstrated for rapid and selective heterogeneity analysis of human IgG subclasses and monoclonal antibodies in complex sample matrices. By comparing with pre-capillary formation of affinity complexes between IgG and protein A, post-capillary affinity detection clearly exhibit greater resolving power for examining IgG microheterogeneity. Affinity complex formation prior to CZE analysis, however, has the advantage of lower detection limits. Detection limits suffer with post-capillary affinity detection because of the high fluorescence background contributed by the fluorescently labeled protein A in the post-capillary reactor, and the need to determine a small change in the background level upon complex formation.     

Characterization of bone protein components with polyacrylamide gel electrophoresis: effects of zinc and hormones in tissue culture

An attempt was made to clarify the molecular characterization of zinc-induced bone protein synthesis in tissue culture. Calvaria were removed from weanling rat (3-week-old male) and cultured for periods up to 48 hr in Dulbecco's Modified Eagle Medium (high Glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. When calvaria cultured in the presence of 10^–5 to 10^–4 M zinc were pulsed with [3H] leucine, zinc caused a significant increase in the incorporation of [³H] leucine into the acid-insoluble residues of bone tissue. The soluble fraction obtained from cultured bone was analyzed with SDS-polyacrylamide gel electrophoresis (SIDS-PAGE). The major components in the fraction obtained from control bone were 68 killo-dalton (kDa) and 45 kDa proteins. These components were clearly increased by the presence of zinc (10^–4 M). The effect of zinc was completely abolished by the coexistence of 10^–6 M cycloheximide. Meanwhile, 10^–9 M estrogen or 10^–8 M insulin, which can stimulate bone formation, did not enhance the effect of zinc to increase bone 68 and 45 kDa proteins. The present findings suggest that zinc increases many bone protein components, especially 68 and 45 kDa proteins.     

Four-Year Changes in Visceral Fat Mass and the Risk of Developing Proteinuria in the General Population

Background

Previous cross-sectional studies demonstrated the close relationship between visceral obesity and the increased prevalence of proteinuria. But, little is known about the role of changes in visceral fat mass (∆VFM) over several years in the development of proteinuria. In this longitudinal cohort study with the general population, the changes in ∆VFM as well as baseline VFM on proteinuria development were evaluated.

Methods

Healthy individuals (n = 2393) who participated in two health screening exams were analyzed. Subjects were divided into three groups based on gender-specific tertiles of baseline VFM and ∆VFM. Each patient was tested for proteinuria using a dipstick, and proteinuria was defined as 1+ or greater.

Results

The mean age was 51.9±7.7 years, and the incidence of proteinuria was 3.9% (n = 93). During the 4 years, 52.5% of the subjects experienced a decline in ∆VFM. However, subjects who developed proteinuria exhibited a significant increase in ∆VFM. Even after adjustment for age, smoking, systolic and diastolic BP, serum creatinine, and hs-CRP levels, the highest tertiles for baseline VFM [men, odds ratio (OR) 3.43, 95% confidence interval (CI) 1.22–9.67; women, OR 2.01, 95% CI 1.05–4.15] and ∆VFM (men, OR 2.92, 95% CI 1.22–6.99; women, OR 3.16, 95% CI 1.56–6.39) were independent predictors of proteinuria development. Following adjustment of both parameters, subjects in the highest baseline VFM and ∆VFM tertiles exhibited the greatest risk of proteinuria development, which suggested the additive harmful effects of the two factors.

Conclusions

Baseline VFM and greater increase in ∆VFM were both important risk factors for developing proteinuria in the general population. Appropriate education and interventions to prevent accumulation of VFM should be the major focus of preemptive strategies.     

Increased noise sensitivity and altered inner ear MENA distribution in VASP−/− mice

Vasodilator-stimulated phosphoprotein (VASP) and mammalian-enabled protein (MENA) share similar cellular localisation and functions (signal transduction pathways, regulation of actin cytoskeleton dynamics). Functional substitution and compensation among Ena/VASP proteins have been proposed as the reason for the absence of major morphological and functional deficits in VASP–/– mice. The aim of this study was to investigate VASP expression in the mouse cochlea, to analyse cochlear function in VASP–/– mice compared with wildtype mice, and to analyse cochlear MENA distribution taking into account that MENA protein might compensate VASP loss in the cochlea of VASP–/– mice. We confirmed specific VASP expression in the pillar cells of the mice organ of Corti as previously reported for rat cochlea. By analysing the hearing function in VASP–/– mice, we found no differences in auditory brainstem responses and distortion product otoacoustic emissions from those of wildtype mice but evidence for an increased noise sensitivity at lower frequencies. When MENA protein levels in cochlea tissue were tested in mutant and wildtype mice by Western blot analysis, no significant differences were found, as was also seen with regard to MENA mRNA levels in laser-microdissected single pillar cells. Most surprisingly, however, MENA protein was absent in pillar cells of VASP–/– mice, whereas it was detected in other cochlear cells. The finding of a cell-specific, and not organ-specific, redundancy of MENA protein expression noted for the first time in VASP–/– mice is proposed as the reason for the observed distinct cochlear phenotype.     

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100–120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1–15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5×10⁴ Cetus units twice daily for 4–5 days), 5-FU augmented (up to 37-fold, days 1–9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3–4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3–9), as determined by limiting-dilution cultures with IL-2 (for 7–8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6–9 days after treatment with 5-FU, Adriamycin or dacarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6–12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1-LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.