Evaluation of Endothelial Dysfunction and Inflammatory Vasculopathy After SARS-CoV-2 Infection-A Cross-Sectional Study.

Background: Rising data suggest that COVID-19 affects vascular endothelium while the underlying mechanisms promoting COVID-19-associated endothelial dysfunction and inflammatory vasculopathy are largely unknown. The aim was to evaluate the contribution of COVID-19 to persisting vascular injury and to identify parameters linked to COVID-19-associated endothelial dysfunction and inflammatory vasculopathy. Methods: In a cross-sectional design, flow-mediated dilation (FMD), nitroglycerine-related dilation (NMD), pulse-wave velocity (PWV), augmentation index, intima-media thickness (IMT), compounds of the arginine and kynurenine metabolism, homocysteine, von Willebrand factor (vWF), endothelial microparticles (EMP), antiendothelial cell antibodies, inflammatory, and immunological parameters, as well as nailfold capillary morphology were measured in post-COVID-19 patients, patients with atherosclerotic cardiovascular diseases (ASCVD) and healthy controls without prior or recent SARS-CoV-2 infection. Results: Post-COVID-19 patients had higher values of PWV, augmentation index, IMT, asymmetric and symmetric dimethylarginine, vWF, homocysteine, CD31+/CD42b- EMP, C-reactive protein, erythrocyte sedimentation rate, interleukin-6, and ß-2-glycoprotein antibodies as well as lower levels of homoarginine and tryptophan compared to healthy controls (all with p < 0.05). A higher total number of pathologically altered inflammatory conditions and higher rates of capillary ramifications, loss, caliber variability, elongations and bushy capillaries with an overall higher microangiopathy evolution score were also observed in post-COVID-19 patients (all with p < 0.05). Most parameters of endothelial dysfunction and inflammation were comparably altered in post-COVID-19 patients and patients with ASCVD, including FMD and NMD. Conclusion: COVID-19 may affect arterial stiffness, capillary morphology, EMP and selected parameters of arginine, kynurenine and homocysteine metabolism as well as of inflammation contributing to COVID-19-associated endothelial dysfunction and inflammatory vasculopathy.

The use and diagnostic value of testing myositis-specific and myositis-associated autoantibodies by line immuno-assay: a retrospective study.

AIMS: Line immune-assays (LIA) for the detection of myositis-specific antibodies (MSA) are used widely for characterization of idiopathic inflammatory myopathies (IIM). Their current use and significance for the diagnosis of IIM remains unclear. METHODS: In this retrospective analysis, we retrieved clinical diagnoses of patients tested for MSA and myositis-associated antibodies (MAA) Jo-1, Mi-2α, Mi-2ß, TIF1γ, SRP, MDA-5, NXP-2, SAE, PL-7, PL-12, EJ, OJ, PM-Scl100, PM-Scl75 and Ku. We calculated clinical specificity, clinical sensitivity, negative- and positive predictive values (PPV) as well as positive and negative likelihood ratios. RESULTS: In total, we analyzed 3167 samples. After exclusion of repeated measurements and patients with insufficient clinical information, data of 1118 patients were available for analysis. A total of 242 patients tested positive for at least one antibody, of which 45 patients had a diagnosis of IIM; 25 IIM patients were negative for all MSA/MAA. Clinical specificity of MSA/MAA for the diagnosis of IIM ranged between 94.2% and 99.9%. Clinical sensitivity and PPV across all antibodies tested ranged from 0.0% to 12.9% and 0.0% to 72.7%, respectively. CONCLUSION: In clinical practice MSA/MAA are used widely for diagnostic work-up of IIM, resulting in a low pre-test probability. Clinicians should be aware that PPVs for most MSA/MAA are low.

Association of Stress Coping Strategies with Immunological Parameters in Melanoma Patients.

In this exploratory case control study the association between stress coping strategies and lymphocyte subpopulations was calculated in 18 non-metastatic melanoma patients and 18 controls with benign skin diseases. Coping strategies were assessed using the German version of the stress-coping questionnaire (SVF 120). While in the control group patients showed significant negative correlations of lymphocyte subpopulations (CD3+, CD4+, CD8+, CD19+, CD45+ cells) with coping strategies that refer to defence, in melanoma patients significant positive correlations between lymphocyte subpopulations (CD3+, CD4+, CD19+, CD45+ cells) were found with regard to coping strategies that are characterized by diversion from stress and focusing on stress-compensating situations. The present data, in melanoma patients and controls, show contrary correlations between stress coping strategies and lymphocyte subpopulations. The interconnection between stress coping and immunologic alterations in malignant melanoma is a field deserving further multiprofessional investigation in order to provide new therapeutical approaches in the treatment and understanding of melanoma patients.

Psychological Stress and Immunological Modulations in Early-stage Melanoma Patients.

Mental stress may have a negative impact on the immune state of cancer patients, in whom immunologic surveillance is essential for survival. This study investigated the immunological response of 19 patients with early-stage melanoma and a matched control group undergoing the Determination Stress Test before surgery. Cytokine and chemokine levels and lymphocyte subpopulations were measured at baseline and post-stress test time-points. Following the stress test lower levels of interleukin (IL)-6 were observed in the melanoma group compared with healthy volunteers (p = 0.044). IL-10 increased significantly in the control group 30 min after the stress test (p = 0.002) in comparison with the melanoma group (p = 0.407). CCL5/Rantes decreased significantly in the melanoma group, whereas CD16/CD56+ natural killer cells increased in both groups, with a sharp decrease below baseline after stress in the melanoma group (p = 0.001). This pilot study shows an altered immunological response to stressors in melanoma patients.

Measurement of tumor necrosis factor-alpha, interleukin-6, Fas ligand, interleukin-1α, and interleukin-1ß in the aqueous humor of patients with open angle glaucoma using multiplex bead analysis.

PURPOSE: Various cytokines, including tumor necrosis factor-alpha (TNF-α), Fas ligand (FasL), interleukin-1α (IL-1α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), contribute to the pathogenesis of primary open angle glaucoma (POAG). The present study was set to measure these cytokines in the aqueous humor of patients with POAG and in control subjects using multiplex bead analysis. METHODS: Twenty-five patients with POAG and 29 control subjects were enrolled in this case-control study. Aqueous humor concentrations of the cytokines (IL-1 α, IL-1 ß, IL-6, FasL, and TNF- α) were measured using multiplex bead analysis. RESULTS: Mean aqueous humor levels of IL-6 were significantly lower in patients with POAG compared to the control subjects (9.3±23.7 versus 55.3±94.4 pg/ml; p=0.002). No significant difference in the aqueous humor concentration of IL-1ß was found between patients with POAG and control subjects (0.5±0.8 versus 0.4±0.8 pg/ml; p=0.85.) Concentrations of IL-1α, TNF-α, and FasL were below limits of detection. No significant correlation was found between IL-6 concentration and age, duration of disease, cup/disc ratio, or mean deviation. CONCLUSIONS: In the present study, we found significantly lower concentrations of IL-6 in the aqueous humor of patients with POAG.

[Laboratory diagnostics for early detection of rheumatic autoimmune diseases: a guide for the general practitioner]. / Labordiagnostik zur Früherkennung und Verlaufskontrolle von systemischen Autoimmunerkrankungen des rheumatischen Formenkreises in der allgemeinmedizinischen Praxis.

Autoimmune diseases are a clinically heterogeneous group of disorders that represent a challenge for the general practitioner in daily routine. Except for rheumatoid arthritis, which is one of the most frequent autoimmune diseases with a prevalence of approximately 1 % of the population, systemic autoimmune disorders are rare. Thus outside specialized wards it might be a challenge to diagnose the underlying autoimmune disease considering the often kaleidoscopic clinical manifestations. Together with careful anamnesis and suspicious clinical symptoms determination of specific autoantibodies can support the suspected diagnosis. The Austrian group of the European autoimmune standardization initiative (EASI) firstly published this guide 2009 with the aim to provide a map through the jungle of the biomarkers for autoimmune diseases for the general practitioner.

Do children and adolescents with type 1 diabetes mellitus have a higher frequency of parietal cell antibodies than healthy controls?

OBJECTIVES: Parietal cell antibodies (PCA) are markers of autoimmune gastritis (AG). AG can lead to hypergastrinemia and iron-deficiency anaemia (IDA). Compared to healthy controls, adults with type 1 diabetes mellitus (T1DM) show a higher prevalence of PCA (1% vs 20%). The aim of the present study was to evaluate the frequency of PCA in children and adolescents with T1DM compared to healthy controls and the clinical and biochemical markers. PATIENTS AND METHODS: We studied 170 patients (87 boys) with T1DM (mean age 12.9 years) and 101 healthy controls (49 boys; mean age 13.0 years). PCA, free T4, free T3, thyroid-stimulating hormone (TSH), and thyroid antibodies were measured in all of the patients. In addition, gastrin, pepsinogen I, iron, ferritin, vitamin B12, and folate were measured in patients with T1DM only. Gastroscopy was carried out in patients with T1DM having high (>100 U/mL) PCA levels. RESULTS: The frequency of PCA in patients with T1DM was 5.29% compared to 1.98% in healthy controls (not significant). PCA was strongly correlated to both thyroid peroxidase antibodies (TPOAb) and gastrin levels (P = 0.001). IDA was present in 4 of 9 patients from the PCA-positive group compared to 4 of 160 patients from the PCA-negative group. Hypergastrinemia was found in 2 PCA-positive patients. Histopathologically, 1 of 4 patients showed early symptoms of AG. CONCLUSIONS: Children and adolescents with T1DM have a lower frequency of PCA than is reported for adults. Compared to healthy controls, they seem to be at increased risk for developing PCA, in particular if positive for TPOAb, but overt clinical disease is rare in children with T1DM.

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. The detection and analysis of such structural modifications may offer a new field for the diagnosis, prognosis and therapy of some diseases. Modern medicine requires new technologies with high sensitivity, specificity and applicability. For the first time in Austria we have combined fluorescence correlation spectroscope (FCS), surface enhanced laser desorption ionisation--time of flight (SELDI-TOF) and the molecular modelling and visualization system according to the computer enhanced programs. Experimental and computational methods are combined in such a way that clinical data can be interpreted by theoretical methods at a molecular level or vice versa, the computational output delivers input for new investigations. One method brings us single results. In view of the spectrum of parameters relevant to clinical entities, multiplexing is a new way of development. Since the technologies are new, the scientifically interested reader should be informed about the matters arising.

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. The designed 'intelligent' probes contained the built-in 5'-fluorescent dyes rhodamine green and Alexa633, respectively, and the 3'-non-fluorescent quenchers BHQ1 and BHQ3, respectively, with perfectly matched spectral overlaps for both dye-quencher combinations. Upon binding of two appropriate probes that were sequence-specific for the genotype, the steady-state fluorescence in two colors increased by about two orders of magnitude. The obtained allele sensitivity of femtomolar and the specificity of the described molecular interactions allow PCR-based allele distinction to be circumvented. Furthermore, the results present an alternative to existing hybridization approaches that are currently used with and without amplification at the 'many-molecule' level and the 'single-molecule' level.

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]. The phylogenetic tree of the human species suggests that there are genetic differences in adaptation patterns to chronic hypoxic hypoxia. Five defense mechanisms are well established for lowlanders who are exposed to acute hypoxic hypoxia. Consequences of the cellular decrease in ATP are the formation of hypoxanthine and xanthine, which are the substrates for the massive formation of superoxide anion radicals and hydrogen peroxide via the oxidase activity of the xanthine oxidoreductase reaction. Under severe hypoxia, about 51 % of the total inhaled oxygen is used to form superoxide anion radicals in rat liver [Gerber et al. (1989) Adv Exp Med Biol 253B, Plenum Press, New York, 497-504]. The reactivity and selectivity of the superoxide anion radical are modified by specific interactions and electron exchange. It is commonly accepted that the superoxide anion radical in aqueous solutions has a lifetime in the millisecond range. However, electron spin resonance spectroscopy studies in a KO2/H2O/iron ion system revealed for the first time a stabilization of a part of the initially added superoxide anion radicals lasting up to hours at room temperature [Földes-Papp (1992) Gen Physiol Biophys 11: 3-38]. Superoxide anion radicals adsorbed on an oxidic iron hydrate phase in aqueous systems might function as a strong oxidant similar to that species which has been suggested to be a complex between oxygen and different valence states of iron in the initiation of lipid peroxidation by ferrous iron. There were serious doubts about the identity of alkoxy radicals. For the first time, alkoxy radicals were directly demonstrated in solution by electron spin resonance spectroscopy [Földes-Papp et al. (1991) Adv Synth Catal 333: 293-301]. The redox status in mammalian cells is mainly determined by the antioxidant glutathione, which is a key player in maintaining the intracellular redox equilibrium and in the metabolic regulation of the cellular defense against oxidative stress. As reactive oxygen species occupy an essential role in membrane damage, the idea of membrane-bound enzymatic defense mechanisms gets a new dimension [Földes-Papp et al. (1981) Acta Biol Med Ger 40: 1129-1132; Földes-Papp and Maretzki (1982) Acta Biol Med Ger 41: 1003-1008]. The steady-state between antioxidants and pro-oxidants affects the gene expression via hypoxia-induced transcription activities. The transcription factor hypoxia-inducible factor 1 (HIF-1) is a global regulator of oxygen homeostasis. As discussed in this article, hypoxia or 'oxidative stress' is accompanied by appropriate molecular adaptation mechanisms at the enzymatic or epigenetic level (enzymatic and non-enzymatic radical inhibitors, posttranslational modifications) and at the genetic level (transcription, translation).

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. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of 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 study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

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. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.

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. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

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. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10(-15) l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small.

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. The molecule consisted of the green-tagged antigen, sandwiched autoantibody and red-tagged secondary (detecting) antibody. A 200-fold increase in sensitivity was obtained as compared to the conventional ELISAs on solid phase. This novel concept has several advantages, namely (i) the sensitivity to detect an individual molecule in solution; (ii) the association of the signal with the reaction event, independent of any immobilization procedure and the artifacts thereof; (iii) the assessment of the broad field of natural antibodies. The theoretical and experimental results obtained bring advanced ultrasensitive analytics to the direct investigation of one and the same single, individual immune molecules as exemplified by the experiments performed with Goodpasture antibody. The novel universal theoretical and experimental framework for continuous measuring the same single, individual immune molecule can be readily transferred to other applications.

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. Mass spectrometry is on its way to the doctor's office (Pusch et al., 2003; Földes-Papp et al., 2002; Henry 1999). The article is focused on laser-activated microprobe mass analysis (LAMMA) and inductively coupled argon plasma mass spectrometry (ICP-MS). Potential applications of the two types of mass spectrometry are demonstrated in clinical medicine. It is the first comprehensive review on qualitative characterization of carbonaceous compounds in lung tissue samples in situ and quantitative trace element determination in body fluids.

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. METHODS: Mass spectrometry was used to evaluate unstained lung sections obtained in vivo from a heavy smoker and a patient with occupationally acquired graphite pneumoconiosis. RESULTS AND DISCUSSION: The composition of carbon compounds deposited in lung tissue samples is demonstrated here for the first time. Thirty carbonaceous-containing microareas from ten biopsies (three areas per biopsy) of lung tissues were analyzed mass-spectrometrically. In each case, the samples were taken from a smoker's lung or those demonstrating a graphite pneumoconiosis. The lung-tissue samples were selected by light microscopy before they were evaporated for mass spectrometry. First-order criteria were anionic and cationic mass peaks which occur within the mass patterns in lung tissues of smoker's lung, although not in graphite pneumoconiosis. Second-order criteria were mass peaks from smoker's lung with standard deviations SD < or = 14% of the mean value. First and second-order mass peaks matched the mass peaks of experimental cigarette-smoke condensate in 9 out of 11 peaks. A software program was developed that enabled fast, automated recognition of the typical mass peaks, and thereby confirmed the histological diagnosis of smoker's lung. CONCLUSIONS: The analysis of carbonaceous particles within lung biopsies from a heavy smoker corresponded to the spectra of tobacco condensate and not to the investigated biopsies of graphite peneumoconiosis. RECOMMENDATION AND OUTLOOK: The analyses were performed in order to find out whether mass-spectrometric criteria exist for the differentiation of carbonaceous lung-tissue deposits. Mass spectrometry may be a valuable tool in determining the composition of carbon compounds deposited in human lung tissue. So far, qualitative assessment of the composition of deposits in lung tissue is only possible after the patient is deceased (autopsy).

Laser scanning confocal fluorescence microscopy: an overview.

Innovative and important aspects of laser scanning confocal fluorescence imaging (LSCFI) are presented here as a general overview. We have described and discussed the technology of the procedure in some detail. We also report some of our original work with transmembranous uptake of 5S gamma-globulin on living human leukocytes as an example of one specific application of LSCFI. These original data and results are presented, as well as citing other uses and applications, to show the power of LSCFI technique. The article will hopefully be useful for those not familiar with the methodology and utility of laser scanning confocal fluorescence microscopy. Applications of LSCFI are very diverse, and there are new applications of this technology constantly being developed. Interest is growing in LSCFI, particularly in the pharmacologic and therapeutic areas, as demonstrated in this article.

C677T single nucleotide polymorphisms of the human methylene tetrahydrofolate reductase and specific identification : a novel strategy using two-color cross-correlation fluorescence spectroscopy.

BACKGROUND: A methylene tetrahydrofolate reductase (MTHFR) deficiency at site C677T renders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency. METHODS: A new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488 nm and 633 nm excitation wavelengths. RESULTS: The measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, the MTHFR genotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis. CONCLUSIONS: We present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target.