Identification of a polygalacturonase (Cup s 2) as the major CCD-bearing allergen in Cupressus sempervirens pollen.

As IgE glyco-epitopes, also referred to as cross-reactive carbohydrate determinants (CCDs), can share significant structural homologies between different plants, they are prone to extensive cross-reactivity among allergen pollen extracts. Here, cypress pollen allergens, especially a polygalacturonase (PG), were further characterized using double one-dimensional electrophoresis (D1-DE). The presence of specific IgE directed against CCDs was investigated by bromelain IgE inhibition and concanavalin A binding assays using sera of cypress pollen-sensitized patients. Our results showed that IgE reactivity to CCDs in Cupressus sempervirens pollen extracts is mainly related to bromelain-type epitopes of a newly identified cypress PG. This glycoprotein has been further characterized through an immunoproteomic approach and officially indexed as Cup s 2 by the WHO/IUIS allergen nomenclature. Cup s 2 could thus be associated with the increased prevalence of IgE reactivity to cypress pollen extracts because of CCD interference.

Complementarity between microarray and immunoblot for the comparative evaluation of IgE repertoire of French and Italian cypress pollen allergic patients.

Cypress pollen represents the primary cause of respiratory allergies in Mediterranean areas. Patients allergic to Cupressus sempervirens pollen (Cups) (CPA) can be discriminated on the basis of the immunoglobulin E (IgE) binding to a basic 14 kDa protein (BP14) or to high-molecular-weight (HMW) glycoproteins only. Specific IgE repertoires of two differentially exposed CPA cohorts, French and Italian, were investigated using an IgE microarray system (some known major allergens from several allergenic sources) and individual IgE immunoblotting (IB) of whole Cups pollen extract separated by SDS-PAGE (all allergens from one allergenic source: cypress pollen). The prevalence of sensitization to BP14 was higher in French (37 %) than in Italian patients (17 %) and major differences were observed in IgE reactivities to lipid transfer proteins (LTPs). Thirty percent of the Italian CPA (4 % in the French group) had specific IgE against the Parietaria pollen LTP, independently of IB subgroups. Regarding peach LTP sensitization, all Pru p 3+ Italian CPA (10 %) were in the HMW+ subgroup, while Pru p 3+ French CPA (20 %) were all included in the BP14+ subgroup. BP14 sensitization is likely a marker of Cups exposure and is, in French CPA, significantly correlated to Pru p 3 sensitization. The IgE immunoblot and microarray are complementary tools that highlight differences in the subtle sensitization profile between groups of patients in comparative studies.

Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

Clostridium difficile O27 colitis: hospital-onset but community-acquired.

Large outbreaks of Clostridium difficile (CD) associated colitis in North America and Europe have been attributed to the emergence of the epidemic/toxin PCR Ribotype O27 CD strain (CD027). Due to the increased virulence of this epidemic strain and its propension for causing outbreaks, we performed a structured risk-assessment approach in order to determine the risks associated with the introduction of this strain within our university hospital. From February 2009 to January 2010, we identified 31 cases of CD027 associated colitis, whereby twenty one (67.7%) had symptoms onset more than 48 hours after admission and were classified as nosocomial. These patients had received wide-spectrum antimicrobials for other infections in the hospital before CD027 colitis diagnosis. The 31 patients with CD027 were admitted in 20 different units, managed by distinct health-care workers (HCWs), and no contact was identified between patients during their hospital stay. Furthermore, infection control audits showed 100% compliance with institutional guidelines for control of CD colitis. These findings suggest that CD027 is most frequently acquired in the community and emerges sporadically under antibiotic pressure during hospitalization.

Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens.

One in five couples of reproductive age has been diagnosed with infertility. Some diagnoses indicate an immunological basis for this disorder. Female immune infertility may be caused by iso-immunization by seminal components. We focused on the characterization of seminal proteins to illustrate the IgG, IgA and IgE immune responses of 31 infertile women. The biochemical characterization was performed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing, both of which were followed by immunoblotting analyses. IgG mainly recognized the antigens with relative molecular masses (Mr) 95 and 183 kDa and isoelectric points ranging from 6.9 to 7.0. The immunodominant antigens recognized by IgA had the Mr of 35 kDa and isoelectric points ranging from 6.2 to 7.2. The reactivity of IgE was not confirmed within our group of patients. The seminal IgG- and IgA -binding patterns were analysed immunochemically to determine the characteristics of possible seminal proteins associated with female immune infertility.

Gibberellin-regulated proteins: Emergent allergens.

About 10 years ago, a protein family was shown for the first time to contain allergenic members, gibberellin-regulated protein (GRP). The first reported member was from peach, Pru p 7. One can hypothesize that it was not detected before because its physicochemical characteristics overlap with those of lipid transfer protein (LTP), a well-known allergen, or because the exposure to GRP increased due to an increase in the gibberellin phythormone level in plant food, either exogenous or endogenous. Like LTPs, GRPs are small cationic proteins with disulfide bridges, are resistant to heat and proteolytic cleavage, and are involved in the defense of the plant. Besides peach, GRP allergens have been described in Japanese apricot (Pru m 7), sweet cherry (Pru av 7), orange (Cit s 7), pomegranate (Pun g 7), bell pepper (Cap a 7), strawberry (Fra a GRP), and also in pollen with a restriction to Cupressaceae tree family (Cup s 7, Cry j 7, and Jun a 7). IgE cross-reactivities were described between GRPs, and the reported peach/cypress and citrus/cypress syndromes may therefore be explained because of these GRP cross-reactivities. GRPs are clinically relevant, and severe adverse reactions may sometimes occur in association with cofactors. More than 60% and up to 95% sequence identities are calculated between various allergenic GRPs, and three-dimensional models show a cleft in the molecule and predict at least three epitopic regions. The structure of the protein and its properties and the matrix effect in the original allergenic source should be unraveled to understand why, despite the ubiquity of the protein family in plants, only a few members are able to sensitize patients.

Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients.

BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.

Patterning of polystyrene by scanning electrochemical microscopy. Biological applications to cell adhesion.

Polystyrene surfaces may be patterned by Ag(II), NO(3)(•), and OH(•) electrogenerated at the tip of a scanning electrochemical microscope. These electrogenerated reagents lead to local surface oxidation of the polymer. The most efficient surface treatment is obtained with Ag(II). The patterns are evidenced by XPS and IR and also by the surface wettability contrast between the hydrophobic virgin surface and the hydrophilic pattern. Such Ag(II) treatment of a polystyrene Petri dish generates discriminative surfaces able to promote or disfavor the adhesion of proteins and also the adhesion and growth of adherent cells. The process is also successfully applied to a cyclo-olefin copolymer and should be suitable to pattern any hydrogenated polymer.

Control of a multi-hospital outbreak of KPC-producing Klebsiella pneumoniae type 2 in France, September to October 2009.

An outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae type 2 was detected in September 2009 in two hospitals in a suburb south of Paris, France. In total, 13 KPC-producing K. pneumoniae type 2 cases (four with infections and nine with digestive-tract colonisations) were identified, including a source case transferred from a Greek hospital. Of the 13 cases, seven were secondary cases associated with use of a contaminated duodenoscope used to examine the source case (attack rate: 41%) and five were secondary cases associated with patient-to-patient transmission in hospital. All isolated strains from the 13 patients: (i) exhibited resistance to all antibiotics except gentamicin and colistin, (ii) were more resistant to ertapenem (minimum inhibitory concentration (MIC) always greater than 4 mg/L) than to imipenem (MIC: 1­8 mg/L, depending on the isolate), (iii) carried the blaKPC-2 and blaSHV12 genes and (iv) had an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern. These cases occurred in three hospitals: some were transferred to four other hospitals. Extended infection control measures implemented in the seven hospitals included: (i) limiting transfer of cases and contact patients to other wards, (ii) cohorting separately cases and contact patients, (iii) reinforcing hand hygiene and contact precautions and (iv) systematic screening of contact patients. Overall, 341 contact patients were screened. A year after the outbreak, no additional case has been identified in these seven hospitals. This outbreak emphasises the importance of rapid identification and notification of emerging highly resistant K. pneumoniae strains in order to implement reinforced control measures.

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Surveillance of occupational blood and body fluid exposures among French healthcare workers in 2004.

OBJECTIVE: To estimate the incidence rate of reported occupational blood and body fluid exposures among French healthcare workers (HCWs). DESIGN: Prospective national follow-up of HCWs from January 1 to December 31, 2004. SETTING: University hospitals, hospitals, clinics, local medical centers, and specialized psychiatric centers were included in the study on a voluntary basis. PARTICIPANTS: At participating medical centers, every reported blood and body fluid exposure was documented by the occupational practitioner in charge of the exposed HCW by use of an anonymous, standardized questionnaire. RESULTS: A total of 375 medical centers (15% of French medical centers, accounting for 29% of hospital beds) reported 13,041 blood and body fluid exposures; of these, 9,396 (72.0%) were needlestick injuries. Blood and body fluid exposures were avoidable in 39.1% of cases (5,091 of 13,020), and 52.2% of percutaneous injuries (4,986 of 9,552) were avoidable (5.9% due to needle recapping). Of 10,656 percutaneous injuries, 22.6% occurred during an injection, 17.9% during blood sampling, and 16.6% during surgery. Of 2,065 splashes, 22.6% occurred during nursing activities, 19.1% during surgery, 14.1% during placement or removal of an intravenous line, and 12.0% during manipulation of a tracheotomy tube. The incidence rates of exposures were 8.9 per 100 hospital beds (95% confidence interval [CI], 8.7-9.0 exposures), 2.2 per 100 full-time-equivalent physicians (95% CI, 2.4-2.6 exposures), and 7.0 per 100 full-time-equivalent nurses (95% CI, 6.8-7.2 exposures). Human immunodeficiency virus serological status was unknown for 2,789 (21.4%) of 13,041 patients who were the source of the blood and body fluid exposures. CONCLUSION: National surveillance networks for blood and body fluid exposures help to better document their characteristics and risk factors and can enhance prevention at participating medical centers.

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

[Consumption of hand-hygiene products in health establishments in the west of France]. / La consommation de produits d'hygiène des mains dans les établissements de soins dans l'interrégion Ouest.

OBJECTIVES: The study had for aim to investigate hand hygiene product use in French hospitals between 2000 and 2003. DESIGN: A questionnaire was sent in 2002 and 2 more in 2003 and 2004 (for 2000 to 2003) requiring data on type of hospital, number of beds, staff members, admissions and patient-day, litres of mild soap, antiseptic soap and alcohol-based rub used and price per litre. Indices were calculated accordingly. RESULTS: 574 hospitals answered over the 4 year period (average 143 per year) representing an average of 50 000 beds/year, 80 000 full-time staff positions, 1.2 million admissions and 16 millions patient-days. The median consumption of mild soap was 3.8 l per bed, 2.7 l per staff member, 2.4 l per 100 admissions, and 10.6 ml per patient-day. The median consumption of antiseptic soap was 1 l per bed, 0.8 l per staff member, 4.8 l per 100 admissions, and 3.2 ml per patient-day. The median consumption of alcohol-based rub (HAS) was 0.3 l per bed, 0.3 l per staff-member, 1.5 l per admission, and 0.9 l per patient-day. Between 2000 and 2003, HAS use significantly increased from 69 to 88% (a relative increase of 31%) and the median consumption increased from 0.5 ml to 1.5 ml per patient-day. 370 fully completed grids gave a number of 7 opportunities per patient-day with less than 1 for HAS. CONCLUSION: The best indicator for an infection control practitioners is the quantity of alcohol-based solution in ml/patient-day and HAS per patient-day is the reference.

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

Sperm-associated and circulating IgA and IgG classes of antibodies recognise different antigens on the human sperm plasma membrane.

IgA and IgG antibodies eluted from the surface of spermatozoa obtained from 11 infertile men were used to analyse the antigens defined by each class of sperm-associated antibodies. An enhanced chemiluminescent Western blot technique was developed to detect the low concentrations of immunoglobulins present in the eluted samples. The same analysis was performed with the sperm membrane-specific antibodies isolated from the sera of 8 of the patients included in the study. Sperm-eluted antibodies reacted with a total of 18 protein bands migrating with molecular masses of between 110 and 18 kDa. Individual antibody-binding patterns differed. Furthermore, IgA and IgG antibodies from any one patient recognised different sets of antigens. In spite of the apparent heterogeneity of the antigens defined by sperm-associated antibodies, the majority of these antibodies reacted with three protein zones of 68/64, 37/36 and 20/18 kDa. The antigens defined by the sperm surface-specific antibodies obtained from the sera of eight infertile patients differed from one patient to another and, in the majority of the patients, differed from those defined by the corresponding sperm-associated antibodies. Nevertheless, two protein zones of 68/64 and 20/18 kDa were recognised by both local and systemic antibodies in 6 and 4 patients, respectively.

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2)).

BACKGROUND: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. METHODS: Timothy grass pollen was exposed to ozone (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) mix. CONCLUSION: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.