Analysis of microdissected prostate tissue with ProteinChip arrays--a way to new insights into carcinogenesis and to diagnostic tools

Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.     

Circulating neutrophil CD14 expression and the inverse association of ambient particulate matter on lung function in asthmatic children.

BACKGROUND: Identifying baseline inflammatory biomarkers that predict susceptibility to size-specific particulate matter (PM) independent of gaseous pollutants could help us better identify asthmatic subpopulations at increased risk for the adverse health effects of PM. OBJECTIVE: To evaluate whether the association between lung function and exposure to ambient levels of PM less than 2.5 microm in diameter (PM2.5) (fine) and 10 to 2.5 microm in diameter (PM(10-2.5)) (coarse) in children with persistent asthma differed across baseline measures of inflammation and innate immune activation. METHODS: We performed a panel study on a local population of 16 children with persistent asthma and evaluated daily pulmonary function (percentage of predicted peak expiratory flow and forced expiratory volume in 1 second) while concurrently measuring daily PM2.5 and PM(10-2.5) exposure from a central site in Chapel Hill, North Carolina. The children underwent a baseline medical evaluation that included assessment of several immunoinflammatory biomarkers in peripheral blood. RESULTS: Children without measurable CD14 expression on circulating neutrophils had significantly reduced pulmonary function (forced expiratory volume in 1 second and peak expiratory flow) with each interquartile range (IQR) increase in PM2.5 (IQR = 8.5 microg/m3) and PM(10-2.5) (IQR = 4.1 microg/m3) concentration, unlike children with measurable CD14 expression (P < .001 for interaction). CONCLUSIONS: Asthmatic children with muted surface expression of CD14 on circulating neutrophils may have a decreased capacity to respond to bacterial components of PM.     

N19 polyepitope as a carrier for enhanced immunogenicity and protective efficacy of meningococcal conjugate vaccines

N19, a string of human universal CD4 T-cell epitopes from various pathogen-derived antigens, was shown to exert a stronger carrier effect than CRM197 for the induction of anti-group C Neisseria meningitidis capsular polysaccharide (MenC), after immunization of mice with various dosages of N19-MenC or CRM-MenC conjugate vaccines. After two immunizations, the N19-based construct induced anti-MenC antibody and protective bactericidal antibody titers higher than those induced by three doses of the CRM-MenC conjugate and required lower amounts of conjugate. N19-based conjugates are superior to CRM-based conjugates to induce protective immune responses to MenC conjugates.     

Recognition of viral antigens in 6/94 virus-induced T-cell-mediated cytotoxicity

Distinct events in the virus-stimulated T-cell-mediated cytotoxicity (V-CMC) have been investigated: 1.) The induction of V-CMC is possible by immunizing mice with infectious as well as UV-inactivated virus (parainfluenza type 1 strain 6/94), or with virus-infected cells either compatible or imcompatible with the recipient. 2.) Recognition of viral antigens by the effector cells occurs independently of the H2 environment: Fractionation of effector cells on columns loaded with virus-infected cells eliminates virus-specific cytotoxic cells. Effector cells and cells on the column need not share H-2 antigens. The findings are discussed with regard to the H2 restriction of the virus induced T-cell mediated cytotoxicity.This paper was presented at the Sixteenth Symposium of the European Association Against Virus Diseases in Amsterdam, September 7–9, 1977     

Differential effects of cancer rehabilitation depending on diagnosis and patients' cognitive coping style

OBJECTIVE: The major aim was to explore the extent to which the Miller Behavioral Style Scale (MBSS) can be used to differentiate cancer patients who are likely to benefit from rehabilitation efforts with a strong information component from those who are not. METHODS: Newly diagnosed patients with breast, gastrointestinal, or prostate cancer (N = 442) were included in a randomized, prospective study of the effects (on anxiety, depression, intrusion, avoidance) of rehabilitation approximately 4 months after diagnosis as compared with control patients. Patients were classified as "monitors" or "blunters" on the basis of the MBSS (368 patients, 83%, completed the MBSS). RESULTS: The expected interaction at postintervention between coping style and experimental condition (ie, rehabilitation or control) was found only for avoidance among breast and prostate cancer patients. Assignment to the rehabilitation or control condition was of no importance for outcome among blunters. Among monitors, the response pattern differed between breast and prostate cancer patients. Prostate cancer monitors seemed to benefit from rehabilitation on all outcome measures, whereas intrusion and avoidance were reduced among breast cancer patients in the control condition. This interaction of diagnosis with condition (rehabilitation or control) among monitors is suggested to be due to demands for diagnosis-specific information during diagnostic work, in the period just after diagnosis, and before treatment decision. CONCLUSIONS: Only the monitor concept seems useful for predicting response to cancer rehabilitation with a strong information component. However, whether rehabilitation is of benefit depends also on other factors.     

Silencing the major apple allergen Mal d 1 by using the RNA interference approach

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.     

Effect of reduced exposure on natural rubber latex sensitization in health care workers.

BACKGROUND: Knowledge about the long-term course of allergy or sensitization to natural rubber latex (NRL) is insufficient. OBJECTIVE: To investigate the long-term effect of preventive measures on sensitization variables in health care workers who had been diagnosed as having NRL allergy (NRLA) or NRL sensitization (NRLS) without clinical symptoms. METHODS: Repeated follow-up investigations, skin prick tests, and NRL specific IgE serum antibodies were performed in 88 health care workers-33 with NRLA and 55 with NRLS. All workers had been instructed to avoid NRL exposure. At the workplace, powder-free NRL gloves for all other employees were gradually introduced. Re-evaluations were done at 14 +/- 3.7 (N = 86) and 38 +/- 4.0 (N = 78) months after the first examination. RESULTS: At the last follow-up, a loss of skin prick test reactivity to NRL was observed in 1 of 29 subjects with NRLA (3.4%) and 16 of 35 with NRLS (45.7%) with previous skin test reactions (P < .001). Among those subjects who demonstrated a kU/L level (CAP class) equal to or greater than class I to NRL at the initial examination, NRL-specific IgE was absent at the last follow-up in 8 (32.0%) of 25 subjects with NRLA and 14 (38.9%) of 36 with NRLS. At the final examination, we could no longer demonstrate sensitization to NRL by any method in 24 (27.3%) of 88 health care workers. Complete loss of NRL sensitization was less frequent in subjects with NRLA than in those with NRLS (1 of 33 or 3.0% vs 23 of 55 or 41.8%; P < .001). CONCLUSIONS: Implementation of simple preventive measures lowers markers of sensitization to NRL quickly in many health care workers with NRLA or NRLS.     

Identification and characterization of linear B-cell epitopes of beta-globulin, a major allergen of sesame seeds

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.     

Requirement for tumor necrosis factor receptor 2 expression on vascular cells to induce experimental cerebral malaria

Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.