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A continuous source for the generation of pure HONO mixtures was developed and characterized, which is based on the Henry's law solubility of HONO in acidic aqueous solutions. With the help of a peristaltic pump, diluted nitrite and sulfuric acid solutions are mixed in a temperature-controlled stripping coil, which is operated with pure nitrogen or synthetic air at gas flow rates of 0.5–2 L min−1. Caused by the acidic conditions of the aqueous phase (pH ≈ 2.5), nitrite is almost completely converted into HONO, which partitions to the gas phase limited by its known solubility in water. The source shows a fast time response of ∼ 2 min (0 %–90 %) at higher concentrations and an excellent long-term stability (2σ noise < 1 %). The HONO emission of the source perfectly correlates with the nitrite concentration from the sub-ppb range up to 500 ppb. The rate of NOx formation increases quadratically with the HONO concentration from non-detectable values at atmospheric relevant HONO concentrations reaching a NOx content of 1.6 % at 500 ppb. A general equation based on Henry's law is developed, whereby the HONO concentration of the source can be calculated using measured experimental parameters, i.e. nitrite concentration, liquid flow rates, gas flow rate, pH of the solution, and temperature of the stripping coil. In the equation, the known Henry's law constant of HONO in sulfuric acid solutions is used. For the calculation of the effective Henry's law constant, the acid dissociation equilibrium of HONO / nitrite is used as a variable to adjust the theoretical HONO concentration to the measured values. From the average of all experimental data the equilibrium of HONO / nitrite is described well by pKa=1021.53/T-0.449. The pKa of 3.0 ± 0.1 (1σ) at 25 ∘C is in good agreement with the range of 2.8–3.28 published in former studies. A standard deviation between all measured and theoretical HONO concentrations of only ±3.8 % was observed, and a conservative upper-limit accuracy of the HONO concentration of better 10 % is estimated. Thus, for the first time, a stable HONO source is developed which can be used for the absolute calibration of HONO instruments.

The last 2 decades have seen substantial technological advances in the development of low-cost air pollution instruments using small sensors. While their use continues to spread across the field of atmospheric chemistry, the air quality monitoring community, and for commercial and private use, challenges remain in ensuring data quality and comparability of calibration methods. This study introduces a seven-step methodology for the field calibration of low-cost sensor systems using reference instrumentation with user-friendly guidelines, open-access code, and a discussion of common barriers to such an approach. The methodology has been developed and is applicable for gas-phase pollutants, such as for the measurement of nitrogen dioxide (NO2) or ozone (O3). A full example of the application of this methodology to a case study in an urban environment using both multiple linear regression (MLR) and the random forest (RF) machine-learning technique is presented with relevant R code provided, including error estimation. In this case, we have applied it to the calibration of metal oxide gas-phase sensors (MOSs). Results reiterate previous findings that MLR and RF are similarly accurate, though with differing limitations. The methodology presented here goes a step further than most studies by including explicit transparent steps for addressing model selection, validation, and tuning, as well as addressing the common issues of autocorrelation and multicollinearity. We also highlight the need for standardized reporting of methods for data cleaning and flagging, model selection and tuning, and model metrics. In the absence of a standardized methodology for the calibration of low-cost sensor systems, we suggest a number of best practices for future studies using low-cost sensor systems to ensure greater comparability of research.

Abstract Background Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. Methods Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100–600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. Results Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 μm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). Conclusions The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016–004547-36).

Reliable measurements of atmospheric trace gases are necessary for both, a better understanding of the chemical processes occurring in the atmosphere, and for the validation of model predictions. Nitrogen dioxide (NO2) is a toxic gas and is thus a regulated air pollutant. Besides, it is of major importance for the oxidation capacity of the atmosphere and plays a pivotal role in the formation of ozone and acid precipitation. Detection of NO2 is a difficult task since many of the different commercial techniques used are affected by interferences. The chemiluminescence instruments that are used for indirect NO2 detection in monitoring networks and smog chambers use either molybdenum or photolytic converters and are affected by either positive (NOy) or negative interferences (radical formation in the photolytic converter). Erroneous conclusions on NO2 can be drawn if these interferences are not taken into consideration. In the present study, NO2 measurements in the urban atmosphere, in a road traffic tunnel and in a smog-chamber using different commercial techniques, i.e. chemiluminescence instruments with molybdenum or photolytic converters, a Luminol based instrument and a new NO2-LOPAP, were compared with spectroscopic techniques, i.e. DOAS and FTIR. Interferences of the different instruments observed during atmospheric measurements were partly characterised in more detail in the smog chamber experiments. Whereas all the commercial instruments showed strong interferences, excellent agreement was obtained between a new NO2-LOPAP instrument and the FTIR technique for the measurements performed in the smog chamber.

A compact and simple instrument for the sensitive detection of NO2 in the atmosphere has been developed. NO2 is sampled in a stripping coil by a selective chemical reaction, converted into a highly absorbing dye, which is detected by long path absorption in a liquid core waveguide. Several interferences were quantified in the laboratory which so far can all be neglected. The significant interferences against HONO and O3 were suppressed by using an upstream HONO/O3-scrubber. The instrument has a detection limit of 2 pptv, an accuracy of 10 % and a precision of 0.5 % for 3 min time resolution. Thus, the new NO2-LOPAP technique is more sensitive than known commercial NO2 instruments. The new analyzer is much simpler to apply than other highly sensitive and selective NO2 methods, e.g. LIF or REMPI methods. The new instrument allows an absolute calibration that can be easily performed with liquid nitrite standards, which is a significant advantage compared with other NO2 measurement techniques for which NO2 calibration gas mixtures are typically necessary. The new instrument has been validated against the chemiluminescence technique during an urban field campaign and against the FTIR technique in a smog chamber under complex photosmog conditions. The data sets exhibit high correlation and excellent agreement.

NO2 concentrations were measured by various instruments during the NO3Comp campaign at the atmosphere simulation chamber SAPHIR at Forschungszentrum Jülich, Germany, in June 2007. Analytical methods included photolytic conversion with chemiluminescence (PC-CLD), broadband cavity ring-down spectroscopy (BBCRDS), pulsed cavity ring-down spectroscopy (CRDS), incoherent broadband cavity-enhanced absorption spectroscopy (IBB\-CEAS), and laser-induced fluorescence (LIF). All broadband absorption spectrometers were optimized for the detection of the main target species of the campaign, NO3, but were also capable of detecting NO2 simultaneously with reduced sensitivity. NO2 mixing ratios in the chamber were within a range characteristic of polluted, urban conditions, with a maximum mixing ratio of approximately 75 ppbv. The overall agreement between measurements of all instruments was excellent. Linear fits of the combined data sets resulted in slopes that differ from unity only within the stated uncertainty of each instrument. Possible interferences from species such as water vapor and ozone were negligible under the experimental conditions.

The oxidation capacity of the highly polluted urban area of Santiago, Chile has been evaluated during a summer measurement campaign carried out from 8–20 March 2005. The hydroxyl (OH) radical budget was evaluated employing a simple quasi-photostationary-state model (PSS) constrained with simultaneous measurements of HONO, HCHO, O3, NO, NO2, j(O1D), j(NO2), 13 alkenes and meteorological parameters. In addition, a zero dimensional photochemical box model based on the Master Chemical Mechanism (MCMv3.1) has been used to estimate production rates and total free radical budgets, including OH, HO2 and RO2. Besides the above parameters, the MCM model has been constrained by the measured CO and volatile organic compounds (VOCs) including alkanes and aromatics. Both models simulate the same OH concentration during daytime indicating that the primary OH sources and sinks included in the simple PSS model predominate. Mixing ratios of the main OH radical precursors were found to be in the range 0.8–7 ppbv (HONO), 0.9–11 ppbv (HCHO) and 0–125 ppbv (O3). The alkenes average mixing ratio was ~58 ppbC accounting for ~12% of the total identified non-methane hydrocarbons (NMHCs). During the daytime (08:00 h–19:00 h), HONO photolysis was shown to be the most important primary OH radical source comprising alone ~55% of the total initial production rate, followed by alkene ozonolysis (~24%) and photolysis of HCHO (~16%) and O3 (~5%). The calculated average and maximum daytime OH production rates from HONO photolysis was 1.7 ppbv h−1 and 3.1 ppbv h−1, respectively. Based on the experimental results a strong photochemical daytime source of HONO is proposed. A detailed analysis of the sources of OH radical precursors has also been carried out.

Background and Aim. The efficiency of tele-monitoring or tele-assistance in patients with severe chronic ventilatory failure in home mechanical ventilation (HMV) is still being investigated. Our aim was to test the feasibility of a model which consisted in: 1) once a week nocturnal telemonitoring, supervised by a doctor in charge in a Respiratory Intensive Care Unit, who also provided a telephone-counselling (24/7) on demand; 2) a scheduled visit every two months. Methods. A 2-year observational study was carried out on 16 patients ventilated for at least 1 year and for ≥ 8 hours /day. Once a week patients underwent a nocturnal monitoring during HMV. The compliance was evaluated by regular transmission of data and regular follow-up, the level of satisfaction by a telephonequestionnaire. Results. The adherence to the protocol study was good in 9/16 (56%) and poor in 7/16 (44%) patients. For each patient, the mean number of connections was 46,12 ± 36.39 (70.7% of that expected), in those with good compliance it increased to 63.8 ± 32.7 (114% of that expected). The median hours of connection was 343 (138- 1019) and 89 (0-521) for patients with good and poor compliance respectively, p=0.038. The mean scheduled visits for patient with good compliance was 6.9 ± 4.14 (100% of that expected). Emergency visits were avoided in 62.5% of cases. The satisfaction score was higher in compliant versus non compliant patients (p=0.019). Conclusion. This pilot study showed that the telemonitoring system employed was feasible and effective in more compliant patients who claimed a high rate of satisfaction.

In this paper we focus on the human arm motion capture, which is motivated by the requirements in physical rehabilitation and training of stroke patients in the same way as monitoring of elderly person activities. The proposed methodology uses a data fusion of low-cost and low-weight MEMS sensors jointly to an a priori knowledge of the arm anatomy. The main goal is to estimate the arm position, the anatomical movements of the shoulder and its accelerations. We propose a discrete optimization based-approach which aims to search the optimal attitude ambiguity directly without decorrelation of ambiguity, and to computing the baseline vector consequently. The originality of this paper is to apply the discrete optimization to track the desired trajectory of a nonlinear system such as the Human Movement in the presence of uncertainties. The global asymptotic convergence of the nonlinear observer is guaranteed. Extensive tests of the presented methodology with real world data illustrate the effectiveness of the proposed procedure.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

This radioimmunoassay for type I collagen mainly detects degradation products of the molecule in human serum samples. Type I collagen antigenicity in serum can be separated into two peaks by gel-filtration chromatography. The larger form represents collagen molecules (as shown by immunoblotting experiments), and (or) type I collagen with aminoterminal propeptide or intact procollagen molecules. The smaller form, the exact nature of which is not known, is quantitatively the principal antigenic form and is derived from degradation of type I collagen. The concentration of type I collagen in serum is increased mainly in cirrhotic patients, with or without active liver disease, but also somewhat in alcoholic patients without cirrhosis.

Section of the anterior cruciate ligament in the knee joint of mature dogs resulted in a progressive fibrillation of both tibial and femoral condylar cartilages. Reduced tissue microhardness and increased water content preceded fibrillation while fixed charge density remained unchanged. A different histological pattern of initial fibrillation was found, however, in femoral and tibial cartilages which exhibited tangential cleft and vertical splits, respectively. Tangential fibrillation alone was associated with necrosis of superficial cells. This suggests that the mechanism of early osteoarthritic cartilage destruction varies in different articular surfaces.

New minor disulfide-bonded collagens were recently described in cartilaginous tissues. The localization of these molecules in epiphyseal proper and growth plate cartilage of fetal calf has been studied using light and electron immunoperoxidase microscopy. The labeling was restricted to the pericellular region of the chondrocytes with an increasing intensity form the superficial to the inner zone of the epiphysis. At the ultrastructural level, the fine-non striated fibrils of the pericellular matrix were stained, demonstrating their collagenous nature. These minor collagenous chains are thus new components of the chondrocyte "exoskeleton" in which other molecules (proteoglycans, chondronectin, fibronectin and type V collagen) have been previously demonstrated.

Section of the anterior cruciate ligament has been performed in the knee of 11 mature dogs. The macroscopically normal cartilage from patella and femoral trochlea of animals killed from 2 to 32 weeks after operation was used for histological, histomorphometrical, and biochemical analysis. Previously undescribed degenerative lesions of the superficial matrix were observed, and there was evidence for secondary healing of these lesions. An early and progressive decrease in superficial cell density and a later progressive increase in cartilage thickness without any change in the cell density of the middle and deep cartilage layers was found. A slight increase in water content with no reduction in glycosaminoglycan content was observed. The results suggest that joint laxity results initially in superficial degenerative changes and later in hypertrophic regenerative changes due to cell proliferation and increased matrix synthesis. Hypertrophic remodeling of articular cartilage in response to abnormal mechanical stresses is postulated.

Summary To detect a foreign collagen in a tissue on a substantially idemtied protein, a radio-tracer technique appeared ideal. The method is only valuable if the introduction of the tram element into the micro molecule does not modify its properties. The first collagen used was tagged by the introduction of C1* into the peptide chain from marked proline by biosynthesis. A 0.3% concentration in sorbic acid solution was applied to the back of the animal and the penetration followed by measuring the activity of tissue sections. No radioactivity was found in the interior of the deinnir after periods of 15, 30,90 and 120 min after application. The method of biosynthetic marking was abandoned as the activity of the protein was considered to be insufficient. The technique of the incorporation of 1(125) in the tyrosine was used to obtain a greater specific activity without modifying the active principle. This chemical treatment resulted from the oxidation of tagged iodide in the presence of collagen. The protein was purified by chromatography and successive dialyses and diluted in unlabelled collagen for subsequent manipulation. Skin applications were made in sorbic acid solution at 0.3% and at 10% in a cosmetic cream. Skin penetration forthe same periods as previously were followed by the same sectionning technique. The penetration reached a maximum at the end of 15 min and was greater for the cream than for the solution. Metabolic studies showed that 15 days after application the residual radioactivity was present mainly in the lymphatic glands and in the skin. Treated dcin showed more activity than untreated areas. At the end of 10 days the excretion of radioactivity had dropped to very low levels. Chromatography on molecular sieve of the collagen extracted from the deeper layers on the skin indicated that the largest part of the radio-activity originated in peptide chains.

We analysed epithelial-mesenchymal interactions that occur during the early stages of the formation of mouse molar roots using light and electron microscopy. Morphological changes observed in the cells of Hertwig's epithelial sheath, the pulp and the follicular mesenchyme are described. The Hertwig's epithelial cells lose their cuboidal form and become flattened, apparently intermixing with the cells of the follicular mesenchyme. At the light- and electron-microscope levels, immunoperoxidase techniques were used to localize fibronectin, laminin and type-IV collagen. These appear to be closely associated with cell differentiation and matrix deposition in developing tooth roots. In addition, at the ultrastructural level, intracellular immunoreactivity was detected. The rough endoplasmic reticulum and nuclear envelope of some cells of the periodontal ligament facing the acellular cementum exhibited specific reactivity with laminin and type-IV collagen. Moreover, these periodontal ligament cells express keratin, but not vimentin, filaments. Our results demonstrate that Hertwig's epithelial cells maintain their capacity to synthesize laminin and type-IV collagen, as well as to express keratin filaments, despite basement membrane fragmentation and the disorganization of Hertwig's epithelial sheath. Thus, some Hertwig's epithelial cells remain in the periodontal ligament intermixed with follicular mesenchyme cells.

The anterior cruciate ligament was severed in the right knee of 36 rabbits which were then killed 12 weeks after surgery. Changes in water content and fixed charge density were correlated with the surface area of induced cartilage fibrillation. Histological changes including cartilage hypertrophy, reduced cell density, matrix alterations, cystic lesions, and enlarged perichondrocytic lacunae were found to precede cartilage fibrillation. The observed histological changes are in agreement with an enzymatic degradation of articular cartilage in osteoarthritis.

A small percentage of patients treated with bovine collagen implants have adverse reactions involving both the cellular and humoral types of immune response. We report a clinical follow-up of 705 subjects treated with a new bovine collagen implant, Atelocollagen (Koken Co. Ltd., Tokyo, Japan). Sensitization to the implant was evaluated in all subjects by skin testing, and humoral response was monitored in 166 subjects by measuring the level of circulating antibodies directed against bovine collagen. Twenty-seven patients (3.8%) exhibited a positive response to a skin test, and of the remaining 656 patients, an adverse reaction to the implant developed in 2.3%. We found a strong correlation between the presence of antibodies to collagen and a positive response to skin testing (92%) or an adverse reaction (100%). In the case of a borderline clinical response to bovine collagen implantation, anticollagen serologic tests appeared to be a useful tool for the identification of clinically reactive patients.

Antibodies to native types I, II, IX, and XI collagen were measured, using a 125I-solid-phase radioimmunoassay, in serum from 104 patients with rheumatic diseases (rheumatoid arthritis, osteoporosis, Paget's disease, or osteoarthritis). In all disease groups, antibodies to type II collagen occurred with greater frequency than antibodies to type I collagen (11-35% versus 5-23%). Antibodies to type XI collagen were the most frequent: They were present in approximately 50% of the patients in the rheumatoid arthritis, Paget's disease, and osteoporosis groups. Antibodies to type IX collagen were found at a high frequency in the rheumatoid arthritis group only (44%). Analysis of the clinical data suggested that the presence of antibodies to collagen was associated with disease that was less severe or of shorter duration.

Cathepsin D antigenicity was localized at the light and electron microscopic levels within dental cells, but not in extracellular matrix. Different intracellular sites for cathepsin D were found depending on the cell type: the enzyme was detected in secretory vesicles of the odontoblasts and in the lysosome-like structures of the ameloblasts. Otherwise, these results suggest that the secretory vesicles of the odontoblasts may contain both cathepsin D and type I collagen. These data might implicate cathepsin D in the enamel and the dentin formations.

The reconstruction of human epidermis during healing of human skin wounded after grafting onto the nude mouse was described in a previous paper (M. Démarchez, P. Sengel, and M. Pruniéras, 1986, Dev. Biol. 113, 90-96). The regeneration of the epidermal basement membrane zone (BMZ) and the reorganization of the connective tissue are the subjects of the present study. They were investigated by two complementary methods: electron microscopy to analyze the BMZ reorganization, and indirect immunofluorescence with species-specific and cross-reacting antibodies directed against laminin, bullous pemphigoid antigen, mouse or human collagens of types I or IV, human elastic fibers, fibronectin, fibrin, actin, and human vimentin, to examine the species origin and distribution of BMZ and connective tissue components during the regeneration process. It is reported that grafted human skin preserves its own immunological markers not only in the epidermis but also in the BMZ and dermis as well, and that, after injury, its regeneration proceeds according to the following sequence of overlapping events: production of a mouse granulation tissue; reepidermization by human cells; reconstruction of a BMZ with human characteristics; formation of a human neodermis. It is concluded that human skin grafted onto the nude mouse is able to regenerate its three structural compartments, namely, the epidermis, BMZ, and dermis. Interestingly, it appeared, also, that the connective tissue regeneration would be a two-step mechanism including the sequential formation of two tissues of distinct sources, namely, a granulation tissue and a neodermis.

Identification of elastic fibers at the ultrastructural level is accomplished by a post-embedding immunohistochemical technique using the protein A-colloidal gold method. Antisera against elastins from human dermis and rat aorta have been characterized by radioimmunoassay and then applied to thin sections of rat blood vessels. Two fixative solutions and two embedding media have been tested. Both antibodies bind to elastic fibers of normal arteries and veins, indicating crossreactions among organs and species. The high sensitivity of this method is demonstrated by its application to the detection of neo-elastogenesis in the intimal thickening of aortic grafts.