Tissue extraction procedures for investigation of urokinase plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in human breast carcinomas.

Urokinase type plasminogen activator (uPA) and its inhibitors, plasminogen activator inhibitor type I (PAI-1) and type II (PAI-2), are supposed to be involved in the expression of the invasive and metastatic phenotype of cancer cells. However, clinical investigations on the prognostic significance of their levels in tumor tissue are difficult to realize because of the absence of a convenient method of measurement of these parameters. The aim of the present investigation was to set up a method allowing the measurement of these enzymes and of sex steroid receptor status in appropriate subcellular fraction(s) in conditions easily reproducible in routine. We found that a tissue homogenate prepared according to the method recommended [5] for current measurement of sex steroid receptors is appropriate for further distinct preparations. One aliquot is used for cytosol preparation; another can be treated by 2% Triton X-100 (vol/vol) and provide an extract containing the totality of uPA and PAI-1. The advantage of this procedure is that appropriate subcellular fractions can be derived from a unique homogenization step. Total uPA and PAI-1 are measured in a Triton extract with good performance as compared to previous investigations [4]. PAI-2 is measured in the same cytosol fraction used for sex steroid receptors and other parameters. Because of its simplicity and its high reliability, this method could be a useful tool in the investigation of uPA family proteases and analysis of their prognostic significance in early breast tumors.

Cross-reactivity between terrestrial snails (Helix species) and house-dust mite (Dermatophagoides pteronyssinus). II. In vitro study.

Epidemiologic and in vitro data have shown that the association of house-dust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection and immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts; molecular weights of snail allergens ranged from < 21 to 200 kDa. Marked individual differences were observed in the seven sera under study; most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin.

Hypersensitivity to banana in latex-allergic patients. Identification of two major banana allergens of 33 and 37 kD.

BACKGROUND: Allergy to banana often occurs in patients sensitized to latex. The spectrum of IgE-mediated responses to latex allergens is more and more documented but banana allergens and epitopes shared by these two allergens have not yet been characterized, even though the existence of cross-reacting IgE antibodies has been demonstrated. OBJECTIVE: The purpose of this study was to assess the relationship between banana hypersensitivity and latex allergy in a population of 19 latex-allergic patients and to identify allergenic components in banana and the common structures shared with latex. METHODS: The in vivo study was conducted in our outpatient department in patients with well-documented latex allergy. Clinical histories were evaluated and skin prick tests were performed with banana and latex extracts. IgE responses from 19 patients were investigated by means of CAP RAST assay and SDS PAGE immunoblotting. Epitopes shared by banana and latex were investigated by means of immunoblotting experiments. RESULTS: Eight of 16 patients (50%) reported symptoms after eating bananas and banana skin prick tests were positive in 5 of 14 patients (36%). Banana RAST results were positive in 12 of the 19 patients (63%). In immunoblot experiments, 17 of the 19 patients (89%) exhibited specific banana IgE antibodies and 16 allergenic components were identified with molecular weights ranging from 17 to 128 kD. Two were considered as major allergens: 33 kD was detectable in 15 of 19 sera (88%) and 37 kD in 13 of 19 sera (76%). Inhibition studies by preincubation of two individual sera with banana or latex extract demonstrated the complete disappearance of IgE binding on banana blotted allergens. CONCLUSION: This study confirms the "latex-fruit syndrome" already described by Blanco et al. Two major allergens were revealed in banana at 33 and 37 kD and the presence of more than ten common components with latex was observed.

Hypersensitivity to latex and Ficus benjamina allergens.

BACKGROUND: Association between allergy to Ficus benjamina and Hevea brasiliensis, two botanically unrelated plants, was suspected in consequence of two clinical observations. Symptoms were rhinitis and asthma. This study was undertaken to assess the in vivo and in vitro cross-reactivity between Ficus benjamina and Hevea brasiliensis allergens. METHODS: The two patients were asked about use and contact with latex devices and relationship between symptoms and Ficus benjamina exposure. Skin prick tests were performed with Ficus benjamina, Hevea brasiliensis extracts and common allergens. Double-blind nasal and bronchial challenge tests were done using the rinse fluid from a brand of latex gloves. Total and specific IgE antibodies to Ficus benjamina and Hevea brasiliensis were determined. In vitro cross-reactivity was investigated by means of CAP RAST and immunodot inhibition experiments. RESULTS: We observed that for the first patient the primary phenomenon is probably allergy to latex followed by allergy to Ficus benjamina. For the second patient, allergy to Ficus benjamina was diagnosed (improvement related to the avoidance of exposure to Ficus benjamina allergens) and positivity to latex skin prick tests may be due to the cross-reacting allergens. In vitro assays showed specific IgE antibodies to both allergens and cross-reactivity was confirmed in the two cases by reciprocal inhibition of the two extracts. CONCLUSION: The increasing risk of sensitization to widely used latex devices and extensive exposure to Ficus species in households and offices indicates increased allergenic risk from this newly recognized cross-reactivity.

Comparison of three commercial kits and a microbiological assay for the determination of vitamin B12 in serum.

Different methods are available for cobalamin determination in serum. Microbiological and radio ligand binding assays are the most commonly used. Kits involving non-isotopic competitive-binding assay have been recently commercialized. In the present work, cobalamins were determined in 146 patient sera, using four methods: a microbiological method, two no boil radio ligand binding assay kits (Magic B12 FOL (NB) from Ciba-Corning and SimulTRAC SNB No Boil from Becton Dickinson) and a non-isotopic kit with acridinium ester labelled cobalamin (Magic Lite from Ciba-Corning). Median (range) cobalamin concentrations in pmol l-1 were 317 (15-1291) using the microbiological method, 355 (25-3469) using the Magic Lite kit, 355 (35-2312) using the Magic B12 FOL (NB) kit and 380 (37-2021) using the SimulTRAC SNB No Boil kit. The ANOVA test indicated that differences between methods were statistically significant (p < 0.01). Competitive-binding methods gave higher results than the microbiological method. Although correlation coefficients were not excellent (0.88 < r < 0.96), the results obtained with the different methods were generally similar and confirmed that competitive methods are useful for detecting low serum concentration of vitamin B12.

The effects of PEG on second antibody immunoprecipitation and its use in immunoassay.

The effects of PEG on the second antibody immunoprecipitation have been studied in several radioimmunological systems. The following parameters have been studied in the assays: the average molecular weight and concentration of PEG, the species of the animal producing the first and second antibody, the nature of the antigen, the concentration of carrier immunoglobulins, and the rate of the immunoprecipitation. Thus, optimal conditions for the use of PEG with a second antibody have been defined in order to be used in any liquid phase immunoassay.

Histamine release assay and radioimmunoassay for the detection of IgE antibodies against neuromuscular blocking drugs.

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anaesthesia. In the present work we evaluated in 41 patients who had experienced anaphylactoid reactions during general anesthesia, the value of different radioimmunoassays (RIAs) and of an histamine release assay (HRA). The RIA was performed with different solid phases as PAPPC (para amino phenyl phosphoryl choline), morphine, TMA (trimethylamine) and TEA (triethylamine). The results were expressed as the percentage of 125I anti-human IgE adsorbed onto the solid phase. The sensitivity was estimated respectively at 95, 82, 93 and 64%. The results were significantly in accordance with those obtained with QAS-RIA (from Guéant) but a weak correlation was seen with Phadebas Rast succinylcholine (r = 0.85 and 0.53 respectively). The HRA gave with the NMBD incriminated a sensitivity of 88%. The correlation between HRA and IDR for the drug involved was 89% and 43% for the other NMBDs. The PAPPC RIA was apparently the most efficient test to screen sera for the presence of IgE antibodies whatever the NMBDs involved in anaphylaxis. An advantage is that this solid phase is commercially available and therefore this RIA can be used routinely with a high sensitivity, allowing the comparison of the results obtained by different laboratories.

Pyridinoline, a mature collagen cross-link, in fibrotic livers from Schistosoma mansoni-infected mice.

The amount of pyridinoline, a cross-linking amino acid found in mature collagen, was measured in murine liver for a period of up to 40 weeks following Schistosoma mansoni infection. The pyridinoline level (expressed as pmol per 100 micrograms of liver) markedly increased in the course of murine schistosomiasis, and the number of pyridinoline residues per collagen molecule was also higher in the livers of infected mice than that of control livers. This increase in the extent of intermolecular collagen cross-linking might play a role in the modulation of collagen catabolism as an auxiliary mechanism of extracellular matrix accumulation during fibrogenesis.

A new radioimmunoassay using a commercially available solid support for the detection of IgE antibodies against muscle relaxants.

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anesthesia. In this study, we developed an in vitro test with a commercially available solid phase for the detection of specific IgE directed to the tertiary or quaternary ammonium groups of neuromuscular-blocking drugs. The solid-phase complex was P-aminophenylphosphoryl-choline (PAPPC) immobilized on agarose, and an RIA was performed with an antihuman IgE labeled with 125I. The results, expressed as the percentage of 125I-labeled anti-IgE linked to the solid phase, were at 0.41 +/- 0.19 for 34 control sera from nonallergic healthy adults, with an upper limit estimated at 1%. The values obtained with the sera of 31 allergic patients ranged from 0.6% to 41% with a sensitivity of 97%. The specificity and the positive predictive value of the PAPPC RIA were 97% and 94%, respectively. These results were compared with results of other RIAs with morphine, trimethylamine, triethylamine immobilized on epoxy-activated Sepharose, and choline hydrochloride immobilized on Sepharose (quaternary ammonium Sepharose RIA) and with Phadebas RAST succinylcholine and Phadebas RAST alcuronium. The PAPPC RIA appears to be the most efficient test to screen sera for the presence of IgE antibodies directed to neuromuscular-blocking drugs. One major advantage is that this solid phase is commercially available and ready to use. This advantage will improve the accuracy in the comparison of the results with results from different laboratories.

Hydroxypyridinium collagen cross-links in human liver fibrosis: study of alveolar echinococcosis.

Liver samples from patients with three different types of liver diseases, alveolar echinococcosis (a dense and irreversible fibrosis), hepatocellular carcinoma and alcoholic cirrhosis, were analyzed for their content in hydroxypyridinium cross-links found in mature collagen. We demonstrated the presence of small amounts of pyridinoline in control livers (0.27 +/- 0.06 pmol/pmol of collagen). Pyridinoline content was increased in fibrotic livers, with the highest values found in patients with alveolar echinococcosis (up to 1.33 pmol/pmol of collagen). The deoxy analogue of pyridinoline was not detected in either normal or fibrotic livers. Pyridinoline levels, expressed as picomoles per picomole of collagen, were similar in all patients with carcinoma (0.7 +/- 0.05 pmol/pmol of collagen). They were heterogeneous in patients with alveolar echinococcosis and were particularly high in patients with alcoholic cirrhosis (1.04 +/- 0.11 pmol/pmol of collagen). These results demonstrate for the first time the presence of an hydroxypyridinium cross-link in liver fibrosis and suggest that pyridinoline measurement might be an important criterion in assessing the irreversibility of human liver fibrosis.

Serum type I collagen and N-terminal peptide of type III procollagen in patients with alcoholic liver disease: relationship to liver histology.

This study compared, in patients with alcoholic liver disease, the serum concentration of N-terminal peptide of type III procollagen and of a novel serum marker, type I collagen, with liver histological data and assessed the role of these markers in the diagnosis and follow-up of liver changes. Ninety-six patients (mean age 51 years, 61 men and 35 women) were included. All had alcoholic liver disease diagnosed on usual clinical, biochemical, and histological criteria. Two histological scores, one for alcoholic hepatitis and one for fibrosis, were established. Serum N-terminal peptide of type III procollagen and type I collagen were assayed by liquid phase radioimmunoassay. Significant correlations between serum type I collagen and score of fibrosis (r = 0.34, p less than 0.001) and between serum N-terminal peptide of type III procollagen and score of alcoholic hepatitis (r = 0.60, p less than 0.0001) were noted. There was no significant correlation between serum aminotransferases and the score of alcoholic hepatitis. In 25 patients with alcoholic hepatitis reassessed between 3 and 6 months, serum N-terminal peptide of type III procollagen significantly decreased (p less than 0.05) as did the score of alcoholic hepatitis, but serum type I collagen and the score of fibrosis were not modified. These serum markers of collagen metabolism could be useful for the assessment and follow-up in patients with alcoholic liver disease.

[Comparison of different methods for serum folate assay]. / Comparaison de différentes méthodes de dosage des folates sériques.

Folates were determined in 148 patient sera, using four different methods: a microbiological assay (Reference Method), two radioassays (Magic B12 FOL [NB] and SimulTRAC SNB no boil kits) and a non isotopic competition method (Magic Lite kit). Folate mean varied according to the methods 10.0 nmol.l-1 (reference method), 12.2 nmol.l-1 (Magic Lite), 8.7 nmol.l-1 (Magic B12 FOL [NB]) and 10.8 nmol.l-1 (SimulTRAC SNB NO boil). Poor correlations were also noted (0.83 < r < 0.95, figures 1 and 2). Differences between methods were explained according to the literature.

Immunoassay techniques for the detection of circulating antibodies to collagen following the use of collagen medical devices.

During the use of collagen medical devices, some adverse clinical reactions occur involving both the cellular and humoral types of the immune response. Thus, the development of immunoassay techniques for measuring the presence and the levels of circulating anticollagen antibodies is required. The authors present their protocol: it is a solid phase radioimmunoassay using collagen coated on polystyrene microplates and labelled protein A as the tracer. An example of the application of the technique is described. Anticollagen antibodies were monitored in 586 patients undergoing bovine collagen implant therapy. In a retrospective study on 166 patients we found a good correlation between the presence of antibodies to collagen and cellular immune reactions such as a positive skin test or adverse clinical reactions after implantation. A prospective study on 420 patients showed that the pretreatment anticollagen serologic test can be useful as an adjunct to skin testing in the conservative management of patients desiring bovine collagen implant therapy. The use of this double test allowed avoidance of any major clinical reaction and reduced minor signs of intolerance.

Aminoterminal propeptide of type III procollagen: a marker of disease activity in schistosomal patients.

The serum concentration of aminoterminal propeptide of type III procollagen was measured in 44 Egyptian healthy controls and 29 patients with hepatosplenomegaly originating from endemic areas for schistosomiasis in Egypt. Patients were classified into two main groups according to the histopathological pattern of the liver biopsy: patients with active schistosomal liver fibrosis and patients with inactive schistosomal liver fibrosis. Serum aminoterminal propeptide of type III procollagen levels were elevated in most of patients with active fibrosis but not in those with inactive schistosomiasis. From the present work, it is suggested that aminoterminal propeptide of type III procollagen can be used as a marker for active fibrogenesis in patients with schistosomal liver fibrosis.

[Vaccination against hepatitis B virus at the Lyon Pasteur Institute. A seven-year evaluation]. / Vaccination contre le virus de l'hépatite B à l'Institut Pasteur de Lyon. Bilan de sept ans.

Results of immunization against hepatitis B among Pasteur Institute staff members are reported. Prior to immunization, 439 subjects were tested for hepatitis B virus (HBV) markers, including HBs antigen, anti-HBs antibody, and anti-HBc antibody (Ausria, Ausab, Corab assays; Abbott). Forty-seven subjects tested positive for anti-HBs antibody. 317 subjects negative for all the HBs markers studied were given three intramuscular doses of Hevac B (Pasteur vaccins) at one-month intervals. Anti-HBs antibodies were assayed after the third injection with the following results: mean titer, 1,454 mIU/ml, standard deviation, 5,349 mIU/ml, and range, 4 to 41,100 mIU/ml. Anti-HBs titers above 10 mIU/ml were found in 879.4% of subjects. Non-responders and weak responders (anti-HBs titer under 10 mIU/ml) were given a fourth dose of vaccine. Ultimately, after the last (third of fourth) injection 97.6% of subjects had protective antibody titers. No case of HBV infection was seen during the seven-year follow-up period.

Serum type I collagen and N-terminal peptide of type III procollagen in chronic hepatitis. Relationship to liver histology and conventional liver tests.

The aim of this study was to compare serum N-terminal peptide of type III procollagen to aminotransferases and gamma-globulins as a marker for histological activity in patients with chronic hepatitis and to assess the role of type I collagen, a new serum marker, as a marker of fibrosis in these patients. Sixty patients with biopsy-proven chronic hepatitis were included in this study. Liver disease was virus B-related in 29, autoimmune in five, drug-induced in five, and of unknown etiology in 21. Each biopsy was independently assessed by two liver pathologists. Two histological scores, a score of activity and a score of fibrosis, were established. Serum N-terminal peptide of type III procollagen and type I collagen were assayed by liquid phase RIA. Significant correlations were noted between serum N-terminal peptide of type III procollagen and scores of activity (r = 0.70, p less than 10(-4)) and fibrosis (r = 0.45, p = 0.0005), and between serum type I collagen and scores of activity (r = 0.46, p = 0.0004) and fibrosis (r = 0.67, p less than 10(-4)). When the correlation between scores of activity and fibrosis (r = 0.52, p = 10(-4)) was considered by partial correlation, serum N-terminal peptide of type III procollagen was correlated with the score of activity (r = 0.63, p less than 10(-3)) but not with the score of fibrosis, and serum type I collagen was correlated with the score of fibrosis (r = 0.58, p less than 10(-3)), but not with the score of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoblotting and cytochemical characterization of human enamel proteins.

Mature enamel proteins (tuft proteins) and fetal enamel proteins were extracted by an homogenizing buffer method, subjected to SDS-PAGE and immunoblotted with a polyclonal antibody raised against the mature enamel proteins. Both fetal and tuft proteins were recognized by this immunoblotting. With the same antibody, immunolocalization of the developing enamel proteins was done on semi-thin-sections of human fetal tissue at the secretory stage, using an immunoperoxidase technique. Specific labelling of the enamel protein matrix was observed. It is concluded that a polyclonal antibody against mature enamel proteins (anti-tuft) can recognize the developing protein matrix at the secretory stage. This suggests that a common antigenic determinant is maintained throughout the course of amelogenesis in human enamel.

Evaluation of automated enzyme immunoassays for several markers for hepatitis A and B using the Abbott IMx analyser.

The analytical performances of the Abbott IMx for the detection of several markers of hepatitis A and B (HBs Ag, anti-HBc, IgM anti-HBc, IgM anti-HAV) were evaluated. The intra-assay and the inter-assay coefficients of variation were below 5 and 10% respectively. We obtained results comparable with our routine methods, except for sera containing rheumatoid factor which gave false positive reactions with the IMx HBs Ag.