Inclusion body myositis in Alzheimer's disease.

BACKGROUND: The prevalence of Alzheimer's disease is increasing. Could findings of similar deposits in brain and muscle tissue explain this increase? The purpose of this report is to illustrate that Alzheimer's disease and inclusion body myositis may share a common aetiology. RESULTS: We present a case where Alzheimer's disease and inclusion body myositis coexist in the same patient. Amyloid-beta deposition and the presence of phosphorylated tau protein have been noted in brain tissue and in muscle biopsy from patients with these disorders. METHODS: Electrophysiological methods are needed for proper diagnosis of this brain and muscle disorder. Recent data on deposit structures in both conditions may indicate an environmental aetiology for Alzheimer's disease and inclusion body myositis. CONCLUSION: By combining electrophysiological methods with muscle biopsy in cases of Alzheimer's disease, the possible aetiological connection between simultaneous affection of both muscle and brain in this condition can be established.

Albumin, transferrin and alpha2-macroglobulin in bronchoalveolar lavage fluid following exposure to organic dust in healthy subjects.

OBJECTIVES: The purpose of the present study was to investigate leakage of plasma proteins in connection with the inflammatory airway reaction following exposure to dust in a pig house. Inhalation of swine-house dust causes intense inflammation with influx of inflammatory cells, predominantly neutrophils, into the airways. The aim of the study was to compare the concentration of three different proteins in bronchoalveolar lavage (BAL) fluid as markers for the inflammation. METHODS: In twenty healthy, non-allergic, non-smokers, not previously exposed to farm dust, BAL was performed approximately 2 weeks before and 24 h after 3 h of exposure to swine dust in a swine-confinement building. Differential cell count and protein concentration were assessed in BAL fluid. Albumin (66.5 kDa) and alpha2-macroglobulin (720 kDa) were quantified by the use of enzyme-linked immunosorbent assay (ELISA) techniques, and transferrin (80 kDa) by zone immunoelectrophoresis assay. The coefficient of variation for repeated protein measurements was <9%. RESULTS: alpha2-Macroglobulin concentration increased six-fold, from 68.0 (36.1-99.9) microg/l, mean (95% CI) before exposure to 411.2 (254.0-568.4) microg/l after exposure (P < 0.001). Transferrin and albumin increased from 19.7 (16.2-23.1) mg/l and 1.8 (1.4-2.2) mg/l, 2.6 and 1.9 times, respectively (P < 0.001). There was significant correlation between the exposure-induced increased protein levels in BAL fluid, although alpha2-macroglobulin was a better discriminator of pre- and post-exposure concentrations than were albumin and transferrin. There was a significant correlation between the exposure-induced BAL-fluid neutrophilia and the increase in alpha2-macroglobulin and transferrin, but not for albumin. This correlation was found only when pre- and post- differences, but not ratios, of plasma proteins were compared. CONCLUSIONS: The levels of plasma proteins increased in BAL fluid following exposure to swine-house dust. alpha2-Macroglobulin was a better marker of this plasma leakage than were albumin and transferrin.

Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase.

It has been established in previous in vitro experiments with human HaCaT keratinocytes that nickel becomes cytotoxic at concentrations higher than 100 microM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of metal insult, i.e. nickel-binding proteins or proteins differentially expressed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) in blots. The cytosolic fraction of cells incubated with nickel, at concentrations over 100 microM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pIs) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. Their pI values corresponded to those of the nickel binding ones seen after IEF. A protein with pI 6.9 had a molecular weight estimated to 38 kDa, two proteins with pI around 7.7 showed molecular weights of 57 and 22 kDa, respectively and another protein with pI of 8.5 had a molecular weight of 33 kDa. The increased abundance of these components, both in IEF experiments and in 2-DE, correlated with the nickel concentration in the culture media. N-terminal amino acid sequencing and database search allowed identification of one a protein as phosphoglycerate kinase and another one as annexin II. The involvement of these proteins in cellular functions and their possible implications in the mechanism of nickel toxicity in keratinocytes are discussed. Some of these proteins may be biomarker candidates for effects of nickel exposure in human keratinocytes.

Airborne birch and grass pollen allergens in street-level shops.

Polluted urban outdoor air may be enriched with large amounts of submicronic respirable pollen allergen particles that penetrate into street-level shops. The objectives of the study were to map concentrations of birch and grass pollen allergens in indoor air of street-level shops and to explore the effect of electrostatic air cleaning under authentic working conditions, indoor air samples were collected in May and July 1999 in two shops. Allergens were quantified by a direct on sampling filter in solution (DOSIS) luminescence immunoassay. The average concentration of airborne indoor birch pollen allergen in the shop with air cleaning was estimated to be 20 +/- 9 SQ/m3 (mean +/- SD) compared to 31 +/- 17 SQ/m3 (mean +/- SD) of that without. The air cleaner reduced the indoor air birch pollen allergen concentration by on average 26 to 48% (P < 0.05). Corresponding figures for airborne indoor grass pollen allergen concentrations were 14 +/- 7 SQ/m3 and 17 +/- 8 SQ/m3, indicating a statistically non-significant (t-test) average 18% reduction of allergen by air cleaning. Excluding two observations with poor fit to the statistical model a significant (P < 0.05) average 27% reduction was obtained. Substantial amounts of airborne birch and grass pollen allergens may occur in street-level shops during flowering seasons.

Miniaturized direct on air sampling filter quantification of pollen allergens.

A recently in this journal reported luminescence immunoassay for the direct quantification of birch and grass pollen allergens on air sampling filters. DOSIS, has been miniaturized. By means of a commercially available chlorinated analogue of the previously used 1,2 dioxetane phosphate derivative as enzyme substrate, the air sampling filter diameter could be reduced from 25 mm to 13 mm. The procedure leads to a more than twenty times reduction of the previously reported limit of quantification for the grass pollen allergen.

Mapping of IgE binding regions in the major rat urinary protein, alpha 2u-globulin, using overlapping peptides.

Exposure to laboratory animals poses a hazard for development of occupational allergy. Identification of antigenic determinants of allergenic proteins may be valuable for immunotherapeutic purposes. Overlapping peptides of the major allergen in rat urine, Rat n 1.02, corresponding to the protein alpha2u-globulin were synthesised on solid support and screened simultaneously to locate IgE binding linear epitopes using a simple modified ELISA procedure. Thirty-nine peptides were synthesised, each 8 amino acids long with 4 amino acids overlaps. Sera from fifteen rat-sensitized subjects were analyzed and as controls sera from 7 non-rat-sensitized individuals were used. In general low binding and a great individual variation between sera from rat allergic individuals were seen. Some peptides were more frequently recognized by IgE antibodies in sera from rat allergics. These peptides were mainly clustered towards the N-terminal and C-terminal parts of the protein. Taken together our data suggest the existence of linear IgE binding epitopes in the rat urine allergen, Rat n 1.02. However, the role of these sequences in the allergic reaction needs further investigation.

Luminescence immunoassay of pollen allergens on air sampling polytetrafluoroethylene filters.

A new method for quantification of airborne birch and grass pollen allergens collected on porous polytetrafluoroethylene filters has been developed. In this method, the allergens firmly adsorbed to the sampling filter of 25 mm in diameter are reacted with specific antibodies conjugated to alkaline phosphatase, generating a matrix-bound allergen-antibody-phosphatase complex. The filter is then floated on a chemiluminescent enzyme substrate solution. The light intensity of the product is linearly related to the amount of allergen over a large mass range, 0-1000 SQ (1 SQ is about 250 pg of protein). This direct on sampling filter in solution (DOSIS) technique demonstrated intra-assay precisions between 6-16% and 11-15% for the levels of 1-100 SQ units of grass allergen Phl p 5 and 4-400 SQ units of birch allergen Bet v 1, respectively. The limits of quantification for the corresponding allergens were estimated to 0.5 and 2 SQ units. Application of DOSIS to analysis of the grass pollen allergen concentrations of outdoor air for 12 days in July 1998 revealed a correlation coefficient of 0.69 between pollen grain and allergen concentrations for the dry weather period. After rainy days large amounts of grass allergens were present even in the absence of pollen grains.

Quantification of birch and grass pollen allergens in indoor air.

Birch and grass pollen grains as well as pollen-derived small particles appear as potent allergens in the outdoor air during spring and summer. The occurrence of pollen allergens in indoor air, however, has not been studied in depth due to lack of suitable sampling and analytical methods. Herein, a recently reported "direct on sampling filter estimation" (DOSAFE) technique (Acevedo et al., 1998) has been validated for quantification of pollen allergens in indoor air using two school rooms and two office rooms as experimental models. Using DOSAFE and polyclonal antibodies against water extracts of pollen from Betula pendula and Phleum pratense L, we found that indoor air of school and office rooms carried substantial amounts of pollen allergens, expressed as SQ units, predominantly occurring as particles with smaller diameters than the pollen grains. In one school room the indoor air birch pollen allergen concentrations increased from 242 to 403 SQ units/m3 over the sampling period although the corresponding outdoor air concentrations decreased from 350 to 90 SQ units/m3. Electrostatic air cleaning in one office room reduced its grass pollen allergen concentrations by more than 95% to 0.02-0.34 SQ units/m3 as compared to the control room.

Visualization and quantification of birch-pollen allergens directly on air-sampling filters.

A new technique is presented to detect and quantify birch (Betula pendula)-pollen allergens directly on air-sampling filters. It is based on the use of specific antibodies, enzymatic reactions, and measurement by chemiluminescence or densitometry. The major pollen antigens are the most important birch allergens. The antibodies used recognize birch-pollen antigens which thus correspond to allergens. Calibration was done with a standardized extract of birch pollen for skin prick testing. The correlation coefficient for the logarithms of luminescence and the amount of birch-pollen allergen applied on filters was > 0.98 in the range 0.04-200 SQ units. A similar correlation was found for the logarithms of the integrated densitometry values and the amount of birch-pollen allergen applied on filters. The number of major pollen-antigen particles or grains on filters could be estimated by counting the major stained spots produced by precipitated enzymatic products. The correlation coefficient was 0.90 for the logarithms of the number of counted major antigen spots and the calculated antigen amount obtained by luminometric measurements. Our results demonstrate that birch-pollen allergens can be determined directly on air-sampling, Teflon-based filters by luminometry, optical density, or particle counting.

In memory of Harry Rilbe: an outstanding scientist who made many valuable contributions to the development of electrophoretic methods.

In July 1997 Professor Harry Rilbe passed away at the age of 84. Harry Rilbe was an outstanding personality skilled in chemistry, optics and mathematics, and he will be remembered for his important contributions to the development of separation methods, notably moving boundary electrophoresis and isoelectric focusing. The latter method is used for analytical and preparative purposes in thousands of laboratories worldwide. Isoelectric focusing is indispensable for characterization of proteins from humans, animals, plants and microorganisms. Isoelectric focusing is thus useful for the understanding of the function of genes and, accordingly, for progress in the life sciences. A biographical retrospect, "A scientific life with chemistry, optics and mathematics", was published by Harry Rilbe in Electrophoresis 1984, 5, 1-17.

Rat urinary proteins binding human IgE detected by chemiluminescence.

Work with laboratory animals involves the increased risk of developing an allergy. Certain proteins in male rat urine have been shown to be major allergens, i.e., Rat n 1.01 and Rat n 1.02. Rat urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were electro-blotted to polyvinylidene difluoride (PVDF) membranes. Immunoblot analysis of human IgE binding components in rat urine utilizing chemiluminescence and a newly developed luminometer is described. Luminometer scanning curves of IgE reactivity to the separated rat urinary proteins are demonstrated. Rat allergic individuals showed different immune responses to the separated proteins. Rat n 1.02, also known as the major protein alpha2mu-globulin, was not always the dominant allergen. Another protein in the albumin region, 60-67 kDa, was found to be an important allergen to some rat-sensitive subjects. Reactivity in the skin prick test with purified Rat n 1.01 and Rat n 1.02 fractions were strong. However, in dot blot under nondenaturing conditions, only weak responses were obtained to the purified rat urinary proteins except for the albumin fraction. Chemiluminescence measurements in blotting membranes of patient IgE bound to different dilutions of certain rat urine proteins revealed good quantitative relationships. Three different chemiluminescence substrates were tested. Measurement of IgE bound to individual allergens as well as the abundance and relative importance of various allergens were studied.

A zone immunoelectrophoresis assay method for quantification of apolipoprotein D in human cerebrospinal fluid.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in human unconcentrated cerebrospinal fluid (CSF). The apo D concentrations of samples of the serum, plasma and CSF were directly proportional to the migration distances of the corresponding zones of immunoprecipitates developed during electrophoresis in glass capillaries filled with antibody-containing agarose gel. A linear standard curve, between about 1 and 12 mg of apo D/1 was obtained using a commercial serum preparation. Seronorm, as apo D standard. The coefficients of variation of the ZIA were below 8% (n = 5 x 6) and 10% (n = 8) for within-run and between-run reproducibility, respectively. Quantification experiments with disulfide-reducing agent, mixtures of CSF and urine as well as frozen and stored CSF samples indicated parallelism between the precipitate-forming immunologic reactions of apo D in different sample matrices when performed with ZIA. Application of this method to quantify apo D of CSF and plasma samples from 51 normal healthy men aged 16-72 years yielded means +/- SD of 5.3 +/- 1.5 mg/l and 128.4 +/- 22.7 mg/l, respectively. No correlation was found between the CSF and plasma apo D concentrations.

A critical review on normal concentrations of vanadium in human blood, serum, and urine.

An evaluation of published values for 'normal' concentrations of vanadium levels in human blood, serum, and urine have been determined in order to identify the reasons for existing large variations of these values. The published data were scrutinized according to criteria on sampling and analysis developed for the TRACY (EUREKA; ENVIRON1) project which aims at establishing reference values for persons without occupational exposure to metals. Of the sampling factors, living in environmentally polluted areas, contamination-free sampling and sample handling were found to be highly important. Expert experience is needed for the accurate vanadium determination in these fluids using well defined radiochemical neutron activation analysis (RNAA) or NAA with pre-irradiation separation or graphite furnace atomic absorption spectrometry (GF-AAS). RNAA is superior for this purpose. Lack of suitable reference materials is a severe handicap in assessing accuracy of vanadium determinations at low levels. Although no reference values for vanadium are established, it appears that values, around 1 nmol l-1 for blood and serum and around 10 nmol l-1 or slightly lower for urine may be considered tentative normal values.

Sample collection guidelines for trace elements in blood and urine. IUPAC Commission of Toxicology.

This paper presents an organized system for element-specific sample collection and handling of human blood (whole blood, serum or plasma, packed cells or erythrocytes) and urine also indicating a proper definition of the subject and sample. Harmonized procedures for collection, preparation, analysis and quality control are suggested. The aim is to assist scientists worldwide to produce comparable data which will be useful on a regional, national and international scale. The guidelines are directed to the elements aluminium, arsenic, cadmium, chromium, cobalt, copper, lead, lithium, manganese, mercury, nickel, selenium and zinc. These include the most important elements measured for their occupational or clinical significance, and serve as examples of principles that will guide development of methods for other elements in the future.

Purification and identification of allergenic alpha (2u)-globulin species of rat urine.

Amino-acid compositional and sequence analyses as well as mass spectrometric determinations of purified rat urine proteins, previously termed prealbumin and alpha(2)-euglobulin, have revealed a high homology between the two forms which have now been identified as alpha(2)-globulin species. The "prealbumin' fraction was found to correspond to alpha(2u)-globulin originating from salivary gland and the 'alpha(2)-euglobulin' fraction was identical with the major urinary protein (MUP) or alpha(2u)-globulin. The results indicate that the two major protein fractions of rat urine constitute different forms of the same parent protein, alpha(2u)-globulin, having no amino-acid sequence resemblance to prealbumin (transthyretin) of rat serum.

Protection against cadmium-metallothionein nephrotoxicity in streptozotocin-induced diabetic rats: role of increased metallothionein synthesis induced by streptozotocin.

Protection against the development of nephrotoxicity following the administration of cadmium-metallothionein (CdMT) at a dose of 0.4 mg Cd per kg body weights was studied in streptozotocin (STZ)-induced diabetic rats. Six groups of Wistar male rats were used (Groups A and B, Groups A1 and C, and Groups A2 and D were injected intraperitoneally with STZ at doses of 0, 50 and 100 mg/kg, respectively, and then 6 days later, Groups B, C and D were injected subcutaneously with CdMT). Proteinuria, albuminuria and transferrinuria were observed after the administration of CdMT, and a dose-related decrease following the increased STZ dose was seen in Groups B, C and D. The concentrations of metallothionein (MT) and zinc (Zn) in liver and kidney were dose-dependently increased in Groups B, C and D. Induction of increased MT synthesis in liver and kidney as the result of the STZ treatment was observed in this study. In particular, a remarkable increase in liver MT concentration was induced by STZ, and transport to the kidney of MT synthesized in liver may perhaps explain the protection against cadmium nephrotoxicity in STZ-induced diabetic rats.

A new luminometer for sensitive quantification by chemiluminescence of specific proteins in microtitre plates and on blot membranes.

Generally applicable technologies are described, which depend on the use of chemiluminescence and a new type of a versatile luminometer, that can measure also weak light in microtitre plates (microplates) and on blotting membranes that is especially useful for dot blot immunoassay. Applications are described using alkaline phosphatase conjugated antibodies against IgG and IgE and the reagent Lumi-Phos 530. This chemiluminescence offers advantages over the use of radioactive isotopes, densitometry and light reflection measurement on membranes and also ELISA, for sensitive quantification of e.g. specific proteins. Special procedures are described for the first time that with the mentioned reagent in agarose gel allows specific and very sensitive quantification of proteins on dot blot membranes. The luminometer, which has temperature control, is very sensitive, precise and allows efficient protocols for various assays. It thus fulfils many of the requirements for good quantification, is time-saving and in addition brings significant improvements due to very low detection limits and large linear concentration ranges that can be measured with excellent regression coefficients (r2 often about 0.999).

Sensitive quantification of proteins by electrophoresis in gels by use of chemiluminescence.

For protein quantitation in gels or blotting membranes, chemiluminescence (CL) offers the advantages of a substantial improvement of detection limits. Easy-to-use CL-chemicals and specific probes such as antibodies conjugated to enzymes, e.g. alkaline phosphatase (AP) may be used in combination with a newly developed luminometer. CL was found to have low detection limits and a linear relation between relative light units (RLU) and the concentration of the antibody enzyme complex present over a wide concentration range. Measurements of the immunoglobulin IgE in dot blots and in blots after sodium dodecyl sulfate (SDS) electrophoresis under nonreducing conditions in agarose gels are described.

Estimation of pooled reference values for cadmium in blood using meta-analysis and TRACY criteria.

Reference values for blood-cadmium levels (B-Cd) are available for only a limited number of geographical areas and for particular population strata (sex, age, smoking habits). This paper, in agreement with the TRACY guidelines, describes and discusses the criteria used to rank published papers on reference values for cadmium retrieved by Medline and Toxline between 1976 and 1991. The TRACY criteria deal with the grading of published papers in terms of their suitability for calculating provisional reference values. Only four out of 18 papers were considered suitable for the TRACY project. The four articles were finally used via meta-analysis to provide provisional reference values for smokers and non-smokers. The comparison of results obtained using published statistics and individual data is used to discuss the appropriateness of meta-analysis in the case of cadmium. Due to the availability of large enough studies and to the clear differences across countries, the suitability of a compound upper reference limit to B-Cd levels seems limited.

Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (ZIA).

For zone immunoelectrophoresis assay (ZIA) glass tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35-150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol-binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.