Enzyme-linked immunosorbent assay (ELISA) for antibodies to Clostridium difficile toxins in patients with pseudomembranous colitis and antibiotic-associated diarrhoea

Enzyme-linked immunosorbent assay (ELISA) was established with purified toxins from Clostridium difficile as antigene to measure antibody response in patiensts with pseudomembranous colitis (PMC) and prolonged antibiotic-associated diarrhoea (AAD). Positive ELISA titres were defined in a control population. Antibodies of IgG class against toxin B were demonstrated in 6/88 (7%) control sera and in 31/61 (51%) sera from 11/19 (58%) patients. Antibodies of IgA class were found in one patient while antibodies of IgM class were not demonstrated. ELISA antibodies against toxin A were not demonstrated. For comparison a neutralization test was performed and neutralizing antibodies to toxin B but not to toxin A were demonstrated in 10/61 (16%) sera from 4/19 (21%) patients and in none of the controls. ELISA was found to be a more sensitive assay than neutralization. ELISA antibodies were detected from the third week of the disease while neutralizing antibodies appeared after 5 weeks. Lack of an antibody response in ELISA seemed to correlate to a more severe colitis.     

The CD94/NKG2 C-type lectin receptor complex

A multigene family of human Ig-SF receptors and members of the murine Ly49 C-type lectin family are involved in natural killer (NK) cell-mediated recognition of MHC class I molecules. The human CD94 glycoprotein covalently assembles with different C-type lectins of the NKG2 family. By functional criteria, the CD94/ NKG2-A (kp43) receptor complex appears also involved in NK cell-mediated recognition of different HLA class I allotypes. Similarly to the other NK inhibitory receptors, NKG2-A contains cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). By contrast, NK clones bearing a different receptor complex (CD94/ p39) are triggered upon ligation by CD94-specific monoclonal antibodies (MAbs); the p39 subunit is likely encoded by other member(s) of the NKG2 family. Expression of the different CD94/ NKG2 complexes is warranted to precisely assess their specific interaction with HLA class I molecules, and the molecular basis for their divergent functional properties.     

Patients'' experiences of GP consultations for psychological problems: a qualitative study

BACKGROUND: The vast majority of patients with psychological problems are seen solely by their GP, but little is known about patients' perspectives regarding the variety of consultation skills that may be used in routine GP consultations with these patients. AIM: To identify which aspects of GP consultations patients presenting with psychological problems experience as helpful or unhelpful. DESIGN: Qualitative study. SETTING: Nine general practices in north central London. METHOD: Twenty patients, who had discussed psychological problems as a significant part of their index GP consultation, were asked in detail using the tape-assisted recall (TAR) method, about aspects of the consultation they had experienced as helpful or unhelpful. RESULTS: All patients described how the relationship with the GP helped or hindered them in discussing their problems; this was central to their experience of the consultation. An underlying attitude of genuine interest and empathy, within a continuing relationship, was highly valued. Patients also described how the GP helped them make sense of, or resolve their problems, and supported their efforts to change. CONCLUSION: These patient accounts suggest that routine GP consultations for psychological problems can have a powerful impact, at least short-term. The GP role in providing a safe place where patients feel they are listened to and understood should not be underestimated, particularly in the mental health context. Further research is required to investigate the longer-term impact of different GP behaviours on patient health outcomes. The TAR method has potential applications in primary care research and in the training of GPs and other health professionals.     

Bruton tyrosine kinase gene mutations in Argentina

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.     

Fluorescence in situ hybridization analysis of HER-2/neu in brushings of normal oral mucosa

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis of HER-2/neu amplification in oral mucosa brushings and to compare the HER-2/neu status with the history and smoking and drinking habits of healthy subjects. Cells obtained by centrifugation of oral brushings from 21 subjects (overall no. of cells: 5125) were suspended in physiological saline and fixed onto two slides for cytological evaluation and FISH analysis (dual-target, dual-color fluorescence assay) of the HER-2/neu gene and CEP17 centromere. A mean of 89.8% of the cells showed two HER-2/neu signals and a mean of 94% had two CEP17 signals at fluorescent microscopy. Finally, a mean of 96% of cells with HER-2/neu / CEP17 had a ratio equal to 1. No association between smoking and drinking habits, age and the HER-2/neu and CEP17 characteristics evaluated by FISH was found.     

Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3

Complement attack is a host strategy leading to elimination of pathogens. Yersinia enterocolitica expresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival of Y. enterocolitica serotype O:3 in nonimmune human serum, we constructed 23 mutant strains of Y. enterocolitica O:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed.     

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.     

Effect of dry cleaning on mite allergen levels in blankets

Since one of the greatest reservoirs of allergens is the blanket, we assessed mite allergen levels in dust collected from five blankets by vacuuming before and after dry cleaning with perchlorethylene and compared the results with five control blankets. Assays with monoclonal antibodies showed that group I ( Der p I and Der f I) mite allergen levels per g dust were 78%, lower after dry cleaning. Group 1 allergen levels per m² of dry-cleaned blankets were 98% lower. RAST inhibition showed that total allergen levels decreased 70% after dry cleaning. Mite allergens were not denatured by perchlorethylene. The effect of dry cleaning resulted from physical washing out of dust and allergens.     

Hox genes specify vertebral types in the presomitic mesoderm

We show here that expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by our finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produced Hox-like phenotypes in the axial skeleton without affecting Hox gene expression.     

Clinically-based determination of safe DNAemia cutoff levels for preemptive therapy or human cytomegalovirus infections in solid organ and hematopoietic stem cell transplant recipients

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.