Molecular diagnosis and prognosis with DNA microarrays.

Microarray analysis makes it possible to determine thousands of gene expression values simultaneously. Changes in gene expression, as a response to diseases, can be detected allowing a better understanding and differentiation of diseases at a molecular level. By comparing different kinds of tissue, for example healthy tissue and cancer tissue, the microarray analysis indicates induced gene activity, repressed gene activity or when there is no change in the gene activity level.

On the occurrence of the diabetes-associated antigen GAD 65 in human sera.

Background: Glutamic acid decarboxylase (GAD 65) is involved as an antigen in diabetes mellitus type 1 and is generally considered to be located intracellularly in pancreas β-cells. In this study we demonstrate its appearance in 64 human sera samples representing a cross-section of a blood bank.

Method: The proof of GAD 65 in sera was done using an enzyme-linked immunosorbent assay (ELISA) setup where it was detected by interaction with corresponding antibodies labeled with an enzyme.

The diabetic antigen glutamic acid decarboxylase (GAD 65) in the human peripheral blood.

Background: Glutamic acid decarboxylase (GAD 65) is a diabetes-associated antigen which is generally considered to be strictly intracellular. In order to better understand autoimmunity, this study demonstrates the appearance of GAD 65 in the peripheral human blood and presents implications for the diagnosis and therapy of some autoimmune diseases.

Methods: The GAD 65 molecules are detected by their interaction with monoclonal antibodies labeled with dyes in an experimental setup with fluorescence correlation spectroscopy (FCS).

The tumour necrosis factor as a mediator of vessel inflammation: importance of exposed receptor residues for its neutralization.

Background: The analysis of the macromolecular tumour necrosis factor (TNF)-receptor interface helps to understand the antigenicity of this inflammatory protein.

Method: The calculations are based on structural data from the protein database. The residues of the macromolecular interface are identified in the interface contact matrix, a plot of pair-wise interactions between adjacent residues in the TNF-receptor complex.

Homology modelling: a review about the method on hand of the diabetic antigen GAD 65 structure prediction.

This introductory paper describes the basic principles and clinical applications of the protein 3D structure prediction by homology modelling. The paper mainly addresses physicians and medical chemists. Because many proteins are of immediate clinical importance, the determination of their structures is crucial for molecular medicine.

Tumour necrosis factor and its receptor: a basic structural analysis of two counterparts.

Inflammation of vessels is partially caused by tumour necrosis factor (TNF). Although the pharmacological understanding of the main inflammatory protein data is well characterised, basic structural information is rare. For this reason, we developed a method for the representation and analysis of the macromolecular interface between TNF and its receptor, enabling a better understanding of their interaction.

DNA microarray analysis: principles and clinical impact.

In recent years, a new technology, allowing the measurements of the expression of thousands of genes simultaneously, has emerged in medicine. This method, called DNA microarray analysis, is today one of the most promising method in functional genomics. Fundamental patterns in gene expression are extracted by several clustering methods like: hierarchical clustering, self organizing maps and support vector machines.

Insights into molecular medicine: development of new diagnostic and prognostic parameters.

Molecular medicine leads us towards an understanding of some diseases at the molecular level. Examples are the analysis of immune complexes and receptor-antireceptor compounds used in clinical medicine. Structural changes of some serum proteins occur in inflammation, neoplasia and autoimmunity.

Influence of lamotrigine and topiramate on MDR1 expression in difficult-to-treat temporal lobe epilepsy.

Purpose: Overexpression of the multiple drug resistance gene 1 (MDR1) was quantified in brain tissue from Coriaria lactone (CL)-kindled Sprague-Dawley (SD) rats after treatment with lamotrigine (LTG) or topiramate (TPM) and compared with that found in rats treated with carbamazepine (CBZ) and valproate (VPA).

Methods: Twenty-five CL-kindled SD rats were randomized into five groups (n = 5 for each group) to receive once-daily feeding of CBZ, VPA, TPM, and LTG as the monotherapy equivalent of maximum human adult dosage, or normal saline (NS control) for 1 month. The expression of P-gp in brain tissues of all rats was quantified by using an image analysis and measuring system (Image Pro-plus 4.

Blockade of tumour necrosis factor {alpha} significantly alters the serum level of IgG- and IgA-rheumatoid factor in patients with rheumatoid arthritis.

Objective: To determine the effect on the humoral immune system of long term treatment of patients with RA with etanercept.

Methods: 12 consecutive patients with seropositive RA treated with etanercept were studied and followed up for 9 months. Clinical efficacy of treatment was evaluated using the 28 joint count Disease Activity Score (DAS28).

[Aspects and necessity of moderate-altitude research].

Alpinism in all its variations is a leading factor in tourism. Within a few decades, alpine sports, even at high altitudes, have become available to a wide range of people. Now, more people than ever before are hiking, trekking, climbing and skiing at moderate and high altitudes.

Oxidative stress caused by acute and chronic exposition to altitude.

In this article, current views on cellular and molecular biology (biochemical) mechanisms are discussed under the aspect of altitude exposition. The Andean, Tibetan, and Ethiopian patterns of adaptation to high-altitude hypoxia are known [Beal et al. (2002) Proc Natl Acad Sci USA 99: 17215-17218].

Lack of specific immunological disease pattern in vulvar lichen sclerosus.

The literature suggests that autoantibody formations and disturbances in cellular or humoral immunities are relevant immunological events in lichen sclerosus (LS). We examined 39 patients (age range: 7-81 years) enrolled in this experimental immunopathology study and treated for vulvar LS. In the serum, we used 88 clinical immunology parameters to evaluate the immunological patterns, i.

A new ultrasensitive way to circumvent PCR-based allele distinction: direct probing of unamplified genomic DNA by solution-phase hybridization using two-color fluorescence cross-correlation spectroscopy.

Single-molecule fluorescence methods enable a new class of nucleic acid assays to be performed that are not possible with PCR-based methods. In this basic study, the methylene tetrahydrofolate reductase (MTHFR)-genotypes (normal, homozygous mutated, as well as heterozygous mutated) were directly detected for the first time onto unamplified double-stranded genomic DNA in solution down to femtomolar allele concentrations (10(-15) M) in a homogeneous assay format. This was accomplished by taking advantage of the decrease by a factor of 40 to 100 in fluorescence background signals of the non-bound nonlinear hybridization probes in two colors and two-color fluorescence cross-correlation spectroscopy.

[Sense and nonsense in antibiotic therapy for respiratory tract infections].

Whether an antibiotic successfully eradicates pathogens depends on the pathogens involved, on pharmacokinetics and bioavailability in the target tissue, and on the antimicrobial resistance of the pathogen. Other determinants are drug interactions, individual risk factors, age and compliance with respect to correct dosage and duration of therapy. In many cases, antimicrobial therapy is begun on an empirical basis, because the responsible pathogen can be identified in only half of all respiratory infections.

Detection of tumour cells in the peripheral blood of patients with breast cancer. Development of a new sensitive and specific immunomolecular assay.

Malignant cells in the peripheral blood of patients with solid tumours are of considerable importance for the prognosis and therapeutic correlation. Their detection however is difficult due to lack of sensitivity, specificity and technical problems in standardisation. In this original article we show a new sensitive method overcoming the hitherto known difficulties by combining traditional antibody-techniques with a RT-PCR.

Clinical interpretation of antineutrophil cytoplasmic antibodies: parvovirus B19 infection as a pitfall.

Background: While antibodies directed against proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) have a high specificity for the diagnosis of systemic vasculitis, they may also be found as an epiphenomenon of acute viral infection.

Objective: To investigate whether positive ANCA test results may be a common feature of acute parvovirus B19 infection.

Methods: Sera were analysed from 1242 patients from a rheumatology outpatient clinic for reactivity with parvovirus B19 and EBV antibodies.

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers.

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types.

Detection of multiple human herpes viruses by DNA microarray technology.

Background: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample.

Methods: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses.

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis.

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes.

A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 2. The individual immune molecule.

In the accompanying original article, the universal theoretical and experimental framework was developed for quantifying one and the same single (selfsame), individual fluorescent-tagged biological molecule without immobilization, hydrodynamic flow or photon burst analysis of fluorescence intensity traces. In the present original article, we describe an application to the detection and identification of circulating anti-glomerular basement membrane antibodies (BMAs) in Goodpasture syndrome. The same single, individual two-color molecule complex was observed among many other molecules.

A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 1. Theory and a first application.

Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many 'different' single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements.

Clinical applicability of mass spectrometry for inhaled carbon compounds and the characterization of trace element patterns in body fluids.

So far, chemists, molecular biologists and biochemists have reaped the greatest benefits from mass spectrometry (Aebersold et al., 2003). This type of analysis could, however, be useful in many fields.

Reconstructed mass-spectrometric pattern for characterization of carbon compounds in smoker's lung in situ.

Background: Cigarette smoke is a major anthropogenic pollutant and contributes to the permanent load of ambient particulate matter in the air, particularly indoors. It is the leading risk factor for premature loss of life due to chronic bronchitis, emphysema and lung cancer. Smoker's lung and graphite pneumoconiosis are pathological states characterized by the deposition of carbonaceous particles.