Biomedical subjects

S H E Kaufmann

Publications and source records attributed to S H E Kaufmann.

8 recordsLinked to original sources

PubMed2006

Deconfounding microarray analysis - independent measurements of cell type proportions used in a regression model to resolve tissue heterogeneity bias.

OBJECTIVES: Microarray analysis requires standardized specimens and evaluation procedures to achieve acceptable results. A major limitation of this method is caused by heterogeneity in the cellular composition of tissue specimens, which frequently confounds data analysis. We introduce a linear model to deconfound gene expression data from tissue heterogeneity for genes exclusively expressed by a single cell type. METHODS: Gene expression data are deconfounded from tissue heterogeneity effects by analyzing them using an appropriate linear regression model. In our illustrating data set tissue heterogeneity is being measured using flow cytometry. Gene expression data are determined in parallel by real time quantitative polymerase chain reaction (qPCR) and microarray analyses. Verification of deconfounding is enabled using protein quantification for the respective marker genes. RESULTS: For our illustrating dataset, quantification of cell type proportions for peripheral blood mononuclear cells (PBMC) from tuberculosis patients and controls revealed differences in B cell and monocyte proportions between both study groups, and thus heterogeneity for the tissue under investigation. Gene expression analyses reflected these differences in celltype distribution. Fitting an appropriate linear model allowed us to deconfound measured transcriptome levels from tissue heterogeneity effects. In the case of monocytes, additional differential expression on the single cell level could be proposed. Protein quantification verified these deconfounded results. CONCLUSIONS: Deconfounding of transcriptome analyses for cellular heterogeneity greatly improves interpretability, and hence the validity of transcriptome profiling results.

Cell Physiological Phenomena↗

PubMed2006

Exploiting immunology and molecular genetics for rational vaccine design against tuberculosis.

One hundred years after the Nobel Prize was awarded to Robert Koch for his work on tuberculosis (TB) and 85 years after the development of the attenuated vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), by Albert Calmette and Camille Guérin, effective prevention measures against TB are still not available. However, the first decade of the 21st century will witness the implementation of clinical trials with several novel vaccine candidates. These candidates fall into two groups: (1) subunit vaccines aimed at boosting the immune response induced by a BCG prime, and (2) recombinant (r)BCG improved to replace the current BCG vaccine strain. For boosting, protein and DNA vaccines in suitable adjuvant or delivery systems, respectively, as well as recombinant viral carriers, such as recombinant modified vaccinia virus Ankara, are being tested. For rBCG prime, a vaccine strain with higher immunogenicity and a strain overexpressing a dominant antigen have been developed. These vaccine candidates will have passed phase I clinical trials before the end of 2006. The goal for the future would be to have these novel vaccine candidates tested in different combinations to facilitate the design of the most efficacious vaccination protocol.

BCG Vaccine↗

PubMed2005

Cationic microparticles consisting of poly(lactide-co-glycolide) and polyethylenimine as carriers systems for parental DNA vaccination.

Cationic microparticles for DNA adsorption were formulated by blending poly(lactide-co-glycolide) (PLGA) (50:50), with different cationic agents, either PEI 25 kDa (polyethylenimine) or CTAB (cetyl-trimethyl-ammonium-bromide). The aim was to create adjuvant delivery systems increasing the efficiency of DNA vaccines. Microparticles formulated with 10% PEI exhibited a highly positive zeta-potential, small particle sizes, in contrast to particles prepared with CTAB, which revealed highly aggregated structures in scanning electron micrographs. PEI 10% microparticles efficiently adsorbed DNA and protected DNA from enzymatic degradation. Microparticles with up to 10% PEI did not affect membrane integrity whereas CTAB particles showed higher LDH release. Transfection efficiencies were assessed using a luciferase reporter gene assay compared to naked DNA and PEI/DNA polyplexes. DNA adsorbed onto microspheres with 10% or 50% PEI generally had higher transfection efficiencies than CTAB but reached lower expression levels than PEI/DNA polyplexes alone. This documented the intact release of DNA. The mechanism of gene delivery to non-phagocytic cells was studied via covalent fluorescence labeling of both the DNA and PEI by confocal microscopy and suggested uptake of DNA. Immunization of mice was performed using plasmids encoding immunodominant antigens of Listeria monocytogenes adsorbed onto RG 502 H+PEI 10% microparticles. The efficiency was tested by intravenous challenge with an otherwise lethal dose of L. monocytogenes. PLGA+PEI microspheres can be used as adjuvant delivery systems for DNA but further optimization is necessary to exploit their full potential.

Adsorption↗

PubMed2005

No life without death--apoptosis as prerequisite for T cell activation.

The orchestrated death of infected cells is key to our understanding of CD8 T cell activation against pathogens. Most intracellular bacteria including Mycobacterium tuberculosis, the etiologic agent of tuberculosis, remain enclosed in phagosomes of infected macrophages. CD8 T cells play a critical role in defense of infection and recognize antigens originating from the cytosol presented by MHC-I molecules. Since mycobacteria do not gain access to the cytosolic MHC-I presentation pathway, the fundamental question as to how CD8 T cells encounter mycobacterial antigens remains to be solved. In this review, we focus on solutions for this enigma and describe the detour pathway of T cell activation. Mycobacteria induce cell death of infected macrophages which thereby leave a last message by releasing apoptotic vesicles. Subsequently, these antigen-containing entities are engulfed by dendritic cells which process the mycobacterial cargo for efficient antigen presentation and CD8 T cell activation. Since the dying infected cell is the origin of a protective T cell response destined to preserve life and individuality, the detour pathway represents an altruistic principle at a cellular level which corresponds to the macroscopic world where death is the precondition to perpetuate the living.

Animals↗

PubMed2005

Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis.

AIMS: To determine the efficacy of room fumigation with vaporized hydrogen peroxide (VHP) in decontamination of viable Mycobacterium tuberculosis. METHODS AND RESULTS: About 8 x 10(4)-2.3 x 10(6) CFU of M. tuberculosis H37Rv and M. tuberculosis Beijing were dried in 10-microl drops in tissue culture plates, placed in steam-permeable Tyvek pouches and distributed on laboratory surfaces. The room was exposed to VHP delivered by air conditioning. Different exposure conditions were tested. Exposure to VHP resulted in sterilization of the bacterial samples in three different test runs. CONCLUSIONS: VHP treatment is an effective means of reducing and eliminating room contaminations of M. tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fumigation with VHP represents an alternative to formaldehyde fumigation, particularly for decontamination of animal rooms in tuberculosis research laboratories.

Anti-Bacterial Agents↗

PubMed2004

New issues in tuberculosis.

Tuberculosis remains a major health problem worldwide. The disease is caused by Mycobacterium tuberculosis whose preferred habitat is the host macrophage. The immune response against tuberculosis is mediated by different subsets of T cells including both conventional CD4 and CD8 T cells as well as unconventional CD1 restricted and gammadelta T cells. The CD1 restricted T cells are particularly remarkable because they recognise the glycolipids abundant in the mycobacterial cell wall. Although a vaccine, M.bovis BCG, is available which protects toddlers against miliary tuberculosis, it is ineffective in preventing pulmonary tuberculosis in adults. Therefore, a novel vaccine is urgently required. Knowledge about the functioning of different T cell populations during infection and disease provides the basis for rational vaccine design. We have constructed a recombinant BCG vaccine which, compared with wild-type BCG, induces superior protection not only against laboratory strains but also against clinical isolates of M. tuberculosis.

BCG Vaccine↗

PubMed2003

[Immunology of tuberculosis: impact on the development of novel vaccines].

Tuberculosis is an ancient health problem that is still not under control worldwide. High infection rates with the etiologic pathogen, Mycobacterium tuberculosis, persisting within the host organism and waiting for the opportunity to disseminate when the immune system is suppressed, and the long and cost-intensive chemotherapeutic treatment urgently require the development of a novel vaccine. This article reviews the immune response to M. tuberculosis infection resulting in new strategies for the improvement of the available vaccine Mycobacterium bovis BCG or for the development of alternative vaccines. A new vaccine should elicit a better immune response than the natural infection and reliably protect from TB disease, regardless if given prior or post infection with M. tuberculosis.

Animals↗

PubMed2002

Protection against tuberculosis: cytokines, T cells, and macrophages.

Tuberculosis remains a major health problem, with two million deaths and eight million new cases annually. At the same time, two billion people (one third of the total world population) are infected with the aetiological agent, Mycobacterium tuberculosis. Of these, fewer than 10% ever develop disease, although the pathogen is not eradicated but rather contained in discrete lesions. Hence, the immune system is highly effective in containing the pathogen, but fails to eradicate it. Disease typically develops through reactivation once the immune system is weakened. The immune response to M tuberculosis is T cell dependent. It comprises not only the conventional CD4 and CD8 T cells, but also gammadelta T cells and CD1 restricted T cells. gammadelta T cells recognise phospholigands and no presentation molecules are known thus far. CD1 restricted T cells recognise glycolipids, which are highly abundant components of the mycobacterial cell wall. Although different T cells are required for optimum protection, the immune mechanisms known to have a role in acquired resistance can be associated with two major mechanisms: (a) activation of macrophages by cytokines; (b) direct cytolytic activity. In vivo granuloma formation, which is central to protection, is induced and sustained by cytokines. Mycobacteria are contained within granulomas and in this way are prevented from spreading all over the body.

Cytokines↗