The influence of allotypes on the IgG subclass response to chromosomal β-lactamase of Pseudomonas aeruginosa in cystic fibrosis patients

Sera from 70 adult cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa were typed for seven GM and two KM allotype determinants. IgG class and all four IgG subclasses of antibodies against chromosomal β-lactamase of Ps. aeruginosa (aβab) were measured in all 70 CF patients in a cross-sectional study. The aβab IgG subclass response in sera collected during the first 11 years of chronic infection from 20 CF patients (10 patients with G3M*5 G1M*3/G3M*5 G1M*3 genotype and 10 patients with G3M*21 G1M*1/G3M*21 G1M*1 genotype) was analysed in a longitudinal study. Increased levels of IgG2 were associated with the presence of GM 23 allotype. IgG3 aβab levels were the lowest for subjects with the GM 1,2,3,17 23 5,21 and GM 1,3,17 21 phenotypes and the highest in subjects with GM 3,23,5 and GM 3,5. No significant differences in IgG1 and IgG4 aβab levels were found between the different phenotypes. IgG1 aβab levels were higher in patients with KM*3/KM*3 genotype compared with patients with KM*3, *1 genotype. Patients with G3M*5 G1M*3/G3M*5 G1M*3 genotype had in both the cross-sectional and the longitudinal study higher IgG3 aβab, lower IgG4 aβab levels and poorer lung function than patients with G3M*21 G1M*1/G3M*21 G1M*1 genotype. An influence of the allotypes on the clinical course of chronic lung infection with Ps. aeruginosa in patients with CF is suggested.     

Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients

Objectives   Typing of Pseudomonas aeruginosa isolates from Norwegian cystic fibrosis (CF) patients with chronic Pseudomonas lung infection in order to see whether cross-infection might have occurred.
Methods   Isolates from 60 patients were collected during the years 1994–98, and typed by pulsed field gel electrophoresis.
Results   Seventy-one strains were identified. One large cluster of identical strains included 27 patients, and 13 smaller clusters of 2–4 patients were found (26 patients). Seven patients had a strain not shared by other patients (private strains). Harboring the main cluster strain was significantly associated with participation in summer camps and training courses ( P  = 0.004, chi-squared test). There were no associations with regular admissions to hospital (intravenous antibiotic courses) or smaller social gatherings of short duration. Small clusters and private strains were not associated with any of the risk factors. All strains were sensitive to colistin. The minimal inhibitory concentrations were generally lower in Norwegian P. aeruginosa strains compared with isolates from Danish patients.
Conclusions   Our results indicate that cross-infection with P. aeruginosa between cystic fibrosis patients has occurred.     

IgG subclass antibodies to Pseudomonas aeruginosa in sera from patients with chronic Ps. aeruginosa infection investigated by ELISA

ELISAs using subclass-specific monoclonal antibodies were developed for the quantification of human IgG1, IgG2, IgG3 and IgG4 antibodies to Ps. aeruginosa. We investigated the pattern of IgG subclass antibodies against Ps. aeruginosa in serum from patients with cystic fibrosis (CF), other patients with chronic Ps. aeruginosa infection, and healthy controls. Healthy controls and patients with CF but without Ps. aeruginosa infection showed no or very low titres of antibodies against Ps. aeruginosa. In the early stage of chronic Ps. aeruginosa infection, antibody titres in all four subclasses were significantly higher than either normals or CF patients without infection. Other patients with Ps. aeruginosa infection showed the same increased level of IgG subclass antibodies as CF patients in an early stage of infection. Sixteen patients (eight in good and eight in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the pre-infection, early and late stages of chronic infection. Patients in a poor clinical condition showed significantly higher levels of IgG3 antibodies in the first year of infection and 2 years later also had significantly higher IgG2 antibody levels. We conclude that elevated levels of IgG2 and IgG3 antibodies to Ps. aeruginosa are a sign of poor prognosis in CF.     

Combined evaluation of circulating immune complexes and antibodies toPseudomonas aeruginosa as an immunologic profile in relation to pulmonary function in cystic fibrosis

We developed a solid-phase radioimmunoassay with a reference standard pseudomonas antigen and used this with¹²⁵I-labeled anti-human immunoglobulin to evaluate specific antibodies toPseudomonas aeruginosa, qualitatively and quantitatively, in sera from children with cystic fibrosis (CF) whose lungs were colonized by this bacterium. The results of this IgG assay correlated with the number of precipitin antibodies to the standard reference antigen determined by cross-immunoelectrophoresis in the same sera. Forced expiratory volume (FEV₁; percentage predicted), determined as an indicator of lung injury in CF, was evaluated as an immunologic response to pseudomonas, against a profile derived from combined serial data on both the circulating immune complexes (CIC) and thePs. aeruginosa antibodies (N=25 CF patients; 108 sera). This revealed that in CF patients who had no specific IgG antibodies toPs. aeruginosa and no IgG-CIC had the best pulmonary function (FEV₁=15±14.52%) and those with high levels of antibodies to this organism and high IgG-CIC levels had the poorest lung function (FEV₁=69.75±10.99%) (P<0.05). We believe that this indicates an immunologic basis for lung injury in cystic fibrosis.     

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6 – 23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis. Received: 25 April 1997     

Pseudomonas aeruginosa strains from the chronically infected cystic fibrosis lung display increased invasiveness of A549 epithelial cells over time

The invasive properties of Pseudomonas aeruginosa pose a serious threat to the wellbeing of cystic fibrosis (CF) patients; however the specific factors affecting invasiveness are not well understood, especially in chronic infection. This study characterises the invasive profiles of sequential isolates of the same P. aeruginosa strain collected five to eight years apart from five chronically infected adult CF patients. Strains from three patients were characterised as unique isolates and from two patients as the Australian Epidemic strain (AES-1) by pulsed field gel electrophoresis. The capacity of these strains to invade the human alveolar A549 cell line was examined. Later isolates were significantly more invasive than earlier counterparts from the same patient. Quantitative real-time PCR and Western blotting showed that the increase in invasiveness over time was independent of ExoS expression and secretion. A link between clonality and invasiveness was also identified, with AES-1 isolates more invasive than unique isolates. These results suggest that despite a reduction in some virulence factors such as the Type-3 Secretion System (T3SS) during chronic infection, a particular strain can become more invasive over time. Defining mechanisms behind the increased invasiveness during chronic infection may help identify new therapeutic targets for CF patients.     

Effective prevention of Pseudomonas aeruginosa cross-infection at a cystic fibrosis centre - results of a 10-year prospective study

Pseudomonas aeruginosa is the major pathogen in chronic lung infections of individuals with cystic fibrosis (CF). Unrelated CF patients may acquire P. aeruginosa from the environment or by cross-infection in the CF setting. We tested the efficacy of measures to prevent nosocomial acquisition of P. aeruginosa at a Paediatric CF centre in a prospective 10-year study. P. aeruginosa-positive and P. aeruginosa-negative patients were seen in alternating weeks at the outpatient clinic. Faucets were equipped with filters to prevent bacterial contamination of tap water. Serial isolates were collected since the first documentation of a P. aeruginosa-positive culture and genotyped with a multimarker microarray. During the 10-year study, the annual prevalence of patients with at least one P. aeruginosa-positive culture was 39±6% in a population of 149±12 patients. P. aeruginosa was detected for the first time in 54 patients of whom 11 patients became chronically colonised with P. aeruginosa. Transient colonisations were recorded 97 times. A nosocomial acquisition of P. aeruginosa at the CF centre probably happened in one case. The worldwide dominant clones in the global P. aeruginosa population were also the most abundant clones in the panel of 324 early CF isolates. No rare clone had expanded by nosocomial transmission. It can be concluded that cross-infection with P. aeruginosa was prevented with simple hygienic measures at a CF centre that had experienced local outbreaks of nosocomial spread among unrelated patients in the past.     

Factors associated with mucoid transition of Pseudomonas aeruginosa in cystic fibrosis patients

Although the mucoid form of Pseudomonas aeruginosa (Pa) is largely responsible for the progression of lung disease in cystic fibrosis (CF), the relationship between factors relating daily-care regimes to mucoidy acquisition are as yet poorly investigated. Fifty-two CF patients registered at the CF centre of Dijon, France, were retrospectively evaluated from the date of Pa colonization either to the first -positive sputum culture for mucoid Pa (n = 26) or to the last culture in which the Pa remained non-mucoid (n = 26). All clinical, pathological and therapeutic events were recorded. The association between the parameters collected and mucoid transition of Pa was assessed in a Cox model with time-dependant covariables. The mean follow-up was 4.7 ± 4.3 years. Three independent parameters were associated with the higher risk of mucoid transition of Pa: persistence of Pa in sputum (OR 7.89; p <0.01), use of inhaled bronchodilators (OR 3.40; p = 0.04), and the use of inhaled colimycin (OR 4.04; p = 0.02). Isolation of Staphylococcus aureus, Haemophilus influenzae or Streptococcus pneumoniae in sputum was associated with a lower risk (OR 0.24; p < 0.01). Mucoid transition of Pa was associated with variables that reflected the severity of both lung disease and Pa colonization. Although they do not lead to prophylactic measures, these results corroborate the need to avoid Pa persistence.     

A genotypic and phenotypic comparison of type III secretion profiles of Pseudomonas aeruginosa cystic fibrosis and bacteremia isolates

The type III secretion system (TTSS) of Pseudomonas aeruginosa enables delivery of a number of toxins involved in the disruption of eukaryotic epithelial surfaces. Whilst the ability to secrete ExoS facilitates invasion and internalization, the secretion of ExoU mediates acute cytotoxicity. In order to determine any association with the ability to secrete these toxins with the nature and severity of human infection, the TTSS genotypes and phenotypes of 163 clinical isolates were determined by multiplex PCR and Western blotting. An exoS+/exoU- genotype was associated with chronic infection in patients with cystic fibrosis whilst an exoS-/exoU+ genotype was associated with strains isolated from blood. Secretion of the ExoU protein was more commonly seen in isolates obtained from blood, suggesting this ability may be important in the development of acute invasive infection. Detection of TTSS toxins in clinical material may be useful in targeting antimicrobial therapy or identifying individuals infected with aggressive strains of P. aeruginosa.     

Polymorphisms in the lectin pathway genes as a possible cause of early chronic Pseudomonas aeruginosa colonization in cystic fibrosis patients

Genes of innate immunity may be involved in early onset of chronic Pa (Pseudomonas aeruginosa) colonization (cPaC) in cystic fibrosis (CF) patients.     

Aberrant IgG subclass distribution to measles in healthy seropositive individuals, in patients with SSPE and in immunoglobulin-deficient patients

Sera from healthy seropositive donors, patients with acute measles, subacute sclerosing panencephalitis, common variable immunodeficiency, and CH gene deletions were analysed for anti-measles IgG1-4. Compared with other anti-viral immune responses of IgG1 and IgG3, an unusual predominance of specific IgG1 prevailed; only four out of a total of 68 patients showed anti-measles IgG3. Of the 17 healthy, measles seropositive serum donors, all showed specific IgG1, none showed IgG3 and six had IgG4. Eight out of 10 patients with SSPE showed an anti-measles IgG1 and IgG4 response while IgG3 was not seen. The IgG1 and IgG4 subclass patterns had some exceptions. Anti-measles IgG3 was found in five out of five patients with deletion of the gamma-1 encoding gene segments and in four out of 15 patients with recent measles antigen stimulation. The subclass pattern was suggested to reflect the immunological compromise associated with measles infections.     

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis

Objective   To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization.
Methods   In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme Spe I. For pyocin typing, the procedure described by Fyfe was applied.
Results   After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig.
Conclusions   Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.     

The predominance of IgG1 and IgG3 subclass antisperm antibodies in infertile patients with serum antisperm antibodies.

Mouse monoclonal antibodies (MAb) specific for each of the four human IgG subclasses and immunofluorescence flow cytometry were used to evaluate the subclass of the IgG antibody response to sperm in serum samples from 13 men and 6 women with a high titer (greater than 1:15,625) of IgG antisperm antibodies (ASA] determined by an indirect immunobead test. Five sera without ASA were also studied as a control. All 19 (100%) of the ASA-positive sera contained immunoglobulin (Ig)G ASA of the IgG1 and IgG3 subclasses. A 1:1 correlation was observed between the presence of IgG1 and IgG3 ASA. IgG2 was essentially undetectable, while IgG4 reactivity, although less intense than IgG1 and IgG3, was more prominent in the sera from the five vasectomized men. The ability of the IgG1 and IgG3 ASA-positive sera to deposit complement (C) on sperm was demonstrated by the concomitant binding to antibody-laden sperm of polyclonal antibodies to the membrane attack complex (C5b-9) of C. Both C-fixing and non-C-fixing ASA-positive sera were found to possess IgG1 and IgG3 antisperm antibodies. The predominance of IgG1 and IgG3 subclasses suggested a T-cell dependent immune response to sperm antigens.     

Titer and subclass distribution of serum IgG antibody reactive withActinobacillus actinomycetemcomitans in localized juvenile periodontitis

Most patients with localized juvenile periodontitis (LJP) manifest serum IgG antibodies specifically reactive with antigens ofActinobacillus actinomycetemcomitans serotype b (Aa-b). Whether these antibodies are protective, destructive, or irrelevant to the progress of the disease remains unclear. We report results of studies aimed at assessing the subclass IgG responses in 35 LJP patients and 35 periodontally normal control subjects using well-characterized monoclonal antibody subclass reagents in an enzyme-linked immunosorbent assay. Our data show that the mean value for total IgG reactive with antigens of Aa-b was more than sevenfold higher for patients than for normal control sera (2349.6 µg/ml for patients vs 332.2 µg/ml for controls). Individual patients and control subjects were classified as high- or low-titer, using twice the median value for total anti-Aa-b IgG in control sera as the cutoff. Of 35 patients, 26 (74%) were high-titer, and 9 (26%) were low-titer. This compares to 5 normal control subjects (14%) high-titer and 30 (86%) low-titer. IgG2 accounted for the major quantitative response in both patients and control subjects. Indeed, the mean IgG2 values for both concentration and percentage of total specific IgG were greater than the combined values for specific anti-Aa-b IgG1, IgG3, and IgG4. Of the 26 high-titer sera, IgG2 predominated in 24, with IgG1 and IgG3 predominating in 1 each; IgG2 predominated in only 2 of the low-titer sera.     

Antimicrobial therapy for pulmonary pathogenic colonisation and infection by Pseudomonas aeruginosa in cystic fibrosis patients

Pseudomonas aeruginosa colonisation has a negative effect on pulmonary function in cystic fibrosis patients. The organism can only be eradicated in the early stage of colonisation, while reduction of bacterial density is desirable during chronic colonisation or exacerbations. Monthly, or at least 3-monthly, microbiological culture is advisable for patients without previous evidence of P. aeruginosa colonisation. Cultures should be performed at least every 2-3 months in patients with well-established colonisation, and always during exacerbations or hospitalisations. Treatment of patients following the first isolation of P. aeruginosa, but with no clinical signs of colonisation, should be with oral ciprofloxacin (15-20 mg/kg twice-daily for 3-4 weeks) plus inhaled tobramycin or colistin (intravenous treatment with or without inhaled treatment can be used as an alternative), while patients with acute infection should be treated for 14-21 days with high doses of two intravenous antimicrobial agents, with or without an inhaled treatment during or at the end of the intravenous treatment. Maintenance treatment after development of chronic P. aeruginosa infection/colonisation (pathogenic colonisation) in stable patients (aged>6 years) should be with inhaled tobramycin (300 mg twice-daily) in 28-day cycles (on-off) or, as an alternative, colistin (1-3 million units twice-daily). Colistin is also a possible choice for patients aged<6 years. Treatment can be completed with oral ciprofloxacin (3-4 weeks every 3-4 months) for patients with mild pulmonary symptoms, or intravenously (every 3-4 months) for those with severe symptoms or isolates with ciprofloxacin resistance. Moderate and serious exacerbations can be treated with intravenous ceftazidime (50-70 mg/kg three-times-daily) or cefepime (50 mg/kg three-times-daily) plus tobramycin (5-10 mg/kg every 24 h) or amikacin (20-30 mg/kg every 24 h) for 2-3 weeks. Oral ciprofloxacin is recommended for patients with mild pulmonary disease. If multiresistant P. aeruginosa is isolated, antimicrobial agents that retain activity are recommended and epidemiological control measures should be established.     

Ig class and IgG subclass responses toTreponema pallidum in patients with syphilis

The Ig class and IgG subclasses of anti-Treponema pallidum antibodies in human serum were quantified using solid-phase enzyme-linked immunosorbent assays. Development of these assays with monoclonal antibodies, each specific for a human immunoglobulin class or IgG subclass, provided quantitative data concerning the major antibody specificities. In patients with primary syphilis, anti-T. pallidum activity was limited almost exclusively to IgG1 and IgM. Coordinate, restricted expression of IgG1 and IgG3 responses inT. pallidum-specific assays was observed with sera from patients with active secondary syphilis. IgG1 and IgG3 accounted for roughly 53 and 43% of the total anti-treponemal IgG antibody activity, respectively. While IgM antibody levels were elevated in the patients with secondary syphilis, IgG2 and IgG4 levels, if present at all, represented less than 10 and 2% of the total IgG activity, respectively. Ig in sera from patients who had been treated adequately for secondary syphilis were restricted almost entirely to IgG3 and IgG1. Considering the low level of IgG3 in serum, disproportionately high percentages of antitreponemal antibodies were found in this subclass during and after treatment for secondary syphilis. The restricted, coexpression of the IgG1 and IgG3 isotypes may reflect the close genetic linkage of the 1 and 3 genes and possibly the impact of immunoregulatory mechanisms in response to the induction and expression of autoantibodies which arise during the course of secondary syphilis.     

Prospective estimation of IgG, IgG subclass and IgE antibodies to dietary proteins in infants with cow milk allergy

Prospectively, serum levels of IgE, specific IgE antibodies (AB) to whole cow milk protein (CMP), bovine se-albumin, bovine immunoglobulin, bovine lactoferrin, bovine lactalbumin and beta-lactoglobulin (BLG), IgG and IgG subclass antibodies to ovalbumin (OA) and BLG, and IgG4 RAST to CMP (bovine whey) were measured in 39 infants with cow milk protein allergy (CMPA) at birth (cord blood), at time of diagnosis and before and after milk challenge at the age of 12 months. Immunological measurements were also undertaken in 33 control infants without CMPA at birth, at 6 months and at 18 months. At no time, were differences found between the levels of IgG and IgG subclass AB to OA and BLG in control versus infants with CMPA. In the 39 infants with CMPA no correlation was found between the levels of IgE, IgG and IgG subclass AB in cord blood and subsequent levels of these values, irrespective of the type of CMPA (IgE-mediated (CMA) or non-IgE-mediated (CMI)), and irrespective of whether remission had occurred. In cord blood 25/33 (76%) of the infants with CMPA had specific IgE-AB to one or more of the bovine milk proteins indicating a prenatal intrauterine sensitization to cow milk protein. At 6 months the frequency of specific IgE-AB to bovine milk proteins was significantly (p less than 0.05) higher in infants with CMA versus CMI, and at 12 months total serum-IgE and the increase of these specific IGE-AB and RAST to CMP were significantly higher (p less than 0.05) in infants with persistent CMA. From 6 to 12 months withholding milk resulted in a significant fall in specific IgE-AB to CMP, and IgG, IgG1 and IgG4 anti-BLG followed by an increase after milk challenge. Decreasing levels of IgG anti-OA from birth to 6 months reflect passive maternal transfer of IgG through the placenta, and increasing levels of IgG anti-BLG, already from birth to 6 months, may represent an early exposure to CMP in all infants. Significantly higher levels (p less than 0.05) of IgG anti-OA AB, IgG1 and IgG4 anti-BLG AB were found in infants with persistent CMA, indicating a close relation between the synthesis of IgE and IgG and between IgE and IgG subclasses (IgG1 and IgG4) in symptomatic cow milk-allergic individuals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pseudomonas aeruginosa isolates from cystic fibrosis patients induce neutrophil extracellular traps with different morphologies that could correlate with their disease severity

Cystic fibrosis (CF) is a disease characterized by bacterial chronic infection of the respiratory tract and inflammation, which leads to a progressive decrease in lung function. Pseudomonas aeruginosa is commonly isolated from the sputum of patients and their presence is associated with a predominant airway inflammation with neutrophils, causing chronic colonization and higher mortality rates. Neutrophil extracellular traps (NETs) have been observed in response against Pseudomonas, however, these cannot eliminate the pathogen from the respiratory tract, so one possibility is that the bacteria could promote their production to use them as a scaffold to colonize the lungs and as a nutrient source, however, their overproduction could also lead to increased damage to the lungs. In this work, we evaluated NETs formation by Pseudomonas clinical isolates obtained from CF patients and found that these induced NETs formation with globular or spread morphologies, of note, we found that there is a trend by which the spread forms were induced mainly by isolates obtained from patients with severe disease, whereas, the globular morphologies were observed for isolates obtained from patients with mild/moderate disease. Finally, we screened for bacterial molecules implicated in NETs formation and found that Exotoxin S, pyocin S2 and pyoverdine could participate in the process.     

Low‐level resistance and clonal diversity of Pseudomonas aeruginosa among chronically colonized cystic fibrosis patients

A prospective study was conducted in Brazil to evaluate antimicrobial resistance patterns and molecular epidemiology of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients with chronic lung infection. All isolates were obtained between May 2009 and June 2010 from 75 patients seen in four reference centers in Brazil: HCPA (20 patients) and HEOM (15 patients), located in southern and northeastern Brazil, respectively; IFF (20 patients) and HUPE (20 patients), both in southwestern Brazil. Antimicrobial susceptibility testing, PCR for detection of carpapenemases, and pulsed‐field gel electrophoresis (PFGE) were performed in 274 isolates. A total of 224 PFGE types were identified and no clones were found circulating among the centers or within the same center. Despite the chronic infection, most patients were colonized by intermittent clones. Only three patients (4%) maintained the same clone during the study. The resistance rates were lower than 30% for the majority of antimicrobials tested in all centers and only 17% of isolates were multiresistant. Isolates (n = 54) with reduced susceptibility to imipenem and/or meropenem presented negative results for bla_SPM‐1, bla_IMP?1, bla_VIM, and bla_KPCgenes. Our results indicate an unexpected low level of antimicrobial resistance and a high genotypic diversity among P. aeruginosa from Brazilian chronic CF patients.