Life-threatening pneumonia caused by human cytomegalovirus and Mycoplasma pneumoniae coinfection in a young, immunocompetent patient.

A young, previously healthy and immunocompetent patient was transferred to our hospital to recover a suspected Ascaris worm from his gall bladder. Although the diagnosis of Ascaris infection could not be confirmed, the patient suffered from cholecystitis. To our surprise, the respiratory situation of the patient deteriorated within 24 h under antibiotic therapy and he had to be transferred to the intensive care unit for mechanical respiration. Human cytomegalovirus (HCMV) was isolated directly from a bronchoalveolar lavage (BAL) sample, and Mycoplasma pneumoniae DNA was detected by PCR in an enrichment culture of the same BAL sample. Serology for HCMV and M. pneumoniae clearly supported a primary/post-primary infection for both agents (IgM detection, increase of IgG titres and, in the case of HCMV, a low avidity index of only 22 %). Therefore, we assumed that a rare HCMV and M. pneumoniae coinfection was the aetiology of the fulminant pneumonia. Under broad antibiotic and antiviral treatment, the situation of the patient improved only very slowly.

Non-tuberculous mycobacterial infection of the parotid gland in an immunosuppressed adult.

Infections of the parotid gland with non-tuberculous mycobacteria (NTM) are rarely described. Here, we report on an infection of the parotid gland caused by Mycobacterium avium and give a literature-based overview about this entity. In the light of a global increase of mycobacterial infections, unusual manifestations have to be considered and should be included in the differential diagnosis when dealing with solid lesions of uncertain aetiology in the head and neck region.

[Quality of life assessment in Inflammatory Bowel Disease (IBD): German version of the Inflammatory Bowel Disease Questionnaire (IBDQ-D; disease-specific instrument for quality of life assessment) -- first application and comparison with international investigations]. / Lebensqualität bei chronisch entzündlichen Darmerkrankungen (CED): die deutsche Version des Inflammatory Bowel Disease Questionnaire (IBDQ-D) zur krankheitsspezifischen Lebensqualitätsmessung -- erste Anwendung und Vergleich mit anderen internationalen Fassungen.

BACKGROUND: Health-related quality of life (HRQOL) is an important outcome-parameter in health research and care. The aim of the working group Quality of Life in the Competence Network Inflammatory Bowel Disease (IBD; in the original German: "Kompetenznetz chronisch entzündliche Darmerkrankungen") is to generate instruments for assessment of HRQOL and its implementation as standards in clinical trials, health care and research in IBD. METHODS: The Inflammatory Bowel Disease Questionnaire (IBDQ) is an international validated disease specific instrument for HRQOL-assessment. A German version of the IBDQ was elaborated and tested in 415 outpatients with Crohn's disease (CD, n = 306) and ulcerative colitis (UC, n = 109). The aim of the study was to compare the results of HRQOL-assessment (IBDQ-D) with international investigations, to correlate HRQOL results with disease activity and to preform a pretest of psychometric properties. RESULTS: International data suggest that the IBDQ-D is a suitable instrument for HRQOL-assessment in CD and UC. For both disease a statistically significant negative correlation with disease activity was found. Tested psychometric properties do not suggest that a revision of the IBDQ-D is required. The IBDQ-D offers the HRQOL-assessment as an primary or secondary outcome in clinical trials in IBD in Germany.

[Erectile dysfunction. Are interdisciplinary diagnosis and therapy necessary?]. / Erektile Dysfunktion. Sind interdisziplinäre Diagnostik und Therapie notwendig?

OBJECTIVE: The last few decades have seen a marked increase in mean life expectancy in Central Europe. This has made elderly people and their quality of life a matter of ever-increasing medical concern. Besides the lack of population based studies in Central Europe, the identifying risk factors for the development of erectile dysfunction (ED) is crucial. METHODS AND MATERIAL: A newly developed and validated questionnaire on male erectile dysfunction was mailed to a representative population sample of 8000 men 30 to 80 years of age in the Cologne urban district. RESULTS: The response included 4489 analysable replies (56,1 %). The median age was 51,8 years. Prevalence of ED was estimated at about 19.2 %, with a steep age-related increase (2,3 - 53,4 %) Therapeutic necessity (defined by co-occurrence of ED and dissatisfaction with sex life), also increases with age. The overall number of ED sufferers seeking therapy was 6,8 %. The following illnesses where seen in the ED group: heart failure (14,7 %), pelvic surgery (18,8 %), diabetes mellitus (20,2 %), peripheral arterial circulatory disorders (21,5 %), herniated disc (23,2 %) and hypertension (32,0 %). Although the pathogenetic pathway remains unclear with a prevalence of 72 % "lower urinary tract symptoms" (LUTS) seems to be an age independent risk factor. In contrast, the prevalence of ED in healthy men was around 6,6 %. CONCLUSIONS: ED is a common disorder, contributing to dissatisfaction with sex life in a considerable proportion of men. ED is frequently associated with chronic diseases. For this reason adequate interdisciplinary diagnostic workup is essential, to offer patients individually adapted treatment.

Implication of therapeutic cloning for organ transplantation.

Replacement of damaged myocardium with electrically functional, contracting syncytium with a balanced blood supply remains a key goal for the treatment of hearts damaged by coronary heart disease or other disorders. Stem cell therapy offers a potential solution. This paper describes the value of in vitro stem cell research to unravel the roles of key regulatory molecules in embryogenesis of myocardium and blood vessels. Studies have shown that functioning myocytes can be derived from stem cells in vitro and engrafted into infarcted areas of heart where they develop into functional adult like cardiomyocytes with action potentials and capacity for beta adrenergic and muscarinic regulation. Further studies have identified specific roles for platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the sequential differentiation of blood vessels and capillaries.

Questions on life satisfaction (FLZM) in inflammatory bowel disease.

BACKGROUND AND AIMS: When assessing quality of care the outcome in terms of quality of life (QOL) is of major significance. This study examined QOL in IBD outpatients and the contribution of individual expectations and various other factors including disease activity. PATIENTS AND METHODS: The study included 306 outpatients with Crohn's disease and 109 with ulcerative colitis (UC). General and health-related QOL was quantified using the instrument Questions on Life Satisfaction(Modules). Disease activity was assessed by a questionnaire. Data were compared with a normal population sample. RESULTS: Life satisfaction scores on general items and on health-related items were significantly lower than in a control sample (60.5+/-37.3 and 74.4+/-41.5, respectively) among both CD patients (54.3+/-33.2, 59.1+/-38.8) and UC patients (45.4+/-34.0, 52.1+/-40.7). Scores were significantly related to severity of disease activity. IBD patients attributed particular importance to health-related issues. CONCLUSION: Both health-related and general life satisfaction is compromised in IBD outpatients, and health-related topics have major impact. Not surprisingly, inflammatory activity compromises QOL, which underlines the importance of anti-inflammatory strategies. The importance attributed to health-related features is higher in IBD patients than in the normal population.

Osteodensitometry of human heel bones by MR spin-echo imaging: comparison with MR gradient-echo imaging and quantitative computed tomography.

The aim of the study was to investigate whether quantitative magnetic resonance (MR) fast spin-echo (FSE) imaging with moderate spatial resolution enables osteodensitometry in peripheral yellow bone marrow. Signal intensities in T1-weighted FSE images from yellow bone marrow indicate the amount of adipose tissue per volume. The signal intensity in marrow regions with spongy bone was assessed and compared to signal intensity of pure fatty marrow (100%). Heel bones of 30 patients with suspected osteoporosis were analyzed and the FSE images were compared with results from parallel MR gradient-echo (GE) imaging and quantitative computed tomography (QCT) examinations. High correlation was found between FSE imaging and QCT [r = 0.91 in the dorsal region of interest (ROI); r = 0.86 in ventral ROI]. Linear correlation coefficients between GE imaging and QCT were slightly lower in the dorsal part (r = -0.86) and considerably lower in the ventral part (r = -0.68). Correlation between the two MR techniques amounted to r = -0.72/-0.61 (dorsal/ventral). The high correlation between FSE imaging and bone mineral density (BMD) allows possible clinical applications of FSE imaging for diagnosis of osteoporosis. Further improvements of the accuracy using reference phantoms might be possible.

Magnetic resonance osteodensitometry in human heel bones: correlation with quantitative computed tomography using different measuring parameters.

RATIONALE AND OBJECTIVES: Density of trabecular bone structures in human heel bones was assessed by 3D magnetic resonance (MR) gradient echo imaging (GEI) with multiple echoes. Different spatial resolutions were applied to investigate the influence of the pixel size on signal characteristics in GEI and to find suitable measuring parameters for a maximum correlation between GEI and bone mineral density obtained by quantitative computed tomography (QCT). METHODS: Thirty-five patients aged 31 to 65 years with suspected osteoporosis underwent MR and QCT examinations of the heel bones. The MR protocol included 3D GEI with three echo times (TE1 = 9.3, TE2 = 27.9, and TE3 = 46.5 ms) and isotropic pixel sizes of (0.6 mm)3, (1.2 mm)3, and (2.4 mm)3. Several subregions in the heel bones were analyzed. For determination of signal reduction with increasing TE, signal intensity ratios were calculated pixelwise from images with TE2/TE1 and TE3/TE1. RESULTS: All examinations showed that the T2*-related signal decrease was more pronounced for lower spatial resolution. In the dorsal part of the heel bones, the correlation between signal ratios in GEI and QCT-based bone mineral density values was between r = -0.86 for a spatial resolution of (0.6 mm)3 and r = -0.73 for (2.4 mm)3. Areas with low trabecular density in the ventral part of the heel bones showed clearly lower correlation coefficients (-0.65 < r < -0.67). CONCLUSIONS: Spatial resolution in 3D GEI clearly influences the T2*-related signal characteristics. Despite measuring different physical properties of spongy bone by GEI and QCT, a relatively high correlation between GEI with small pixel sizes and QCT was obtained in the dorsal part of the heel bones, but not in the ventral part with partly thickened trabeculae and irregular distribution. However, standardized measuring protocols with preferably small pixel sizes (as low as [0.6 mm]3) should be applied, and correlation curves must be determined, dependent on the actual bone marrow site, before clinical routine MR osteodensitometry becomes possible.

Is glutamine essential for the maintenance of intestinal function? A study in the isolated perfused rat small intestine.

Glutamine has received considerable interest as a gut-targeted nutrient due to its proposed key role in the maintenance of intestinal structure and function. We used a preparation of isolated vascularly perfused rat small intestine to investigate whether glutamine is essential for the maintenance of intestinal function. When glutamine was available, arterial glutamine was extracted at 15 +/- 2%, and net uptake was -89 +/- 5 nmol min-1 g-1. Nitrogenous metabolites ammonia, alanine, and citrulline (41 +/- 7, 41 +/- 4, and 11 +/- 2 nmol min-1 g-1, respectively) were released into the venous perfusate, but only ammonia was also excreted into the lumen (36 +/- 3 nmol min-1 g-1). In the absence of exogenous glutamine alanine release was halved and that of citrulline and ammonia nullified. Additional inhibition of glutamine synthetase yielded the same results. In all cases variables of tissue function were fully maintained also in the absence of exogenous and/or endogenous glutamine. The inhibition of glutaminase/amidotransferase reactions, however, was accompanied by a reduction in glutamine consumption and a graded deterioration in tissue function. In conclusion, glutamine seems to be dispensable as a metabolic fuel to be fully oxidized by the mucosa. However, the inhibition of major glutamine consuming pathways was associated with impaired tissue function and viability. Therefore the role of intestinal glutamine metabolism seems to be threefold: (a) providing affluent amounts of nitrogen precursors for mucosal anabolic pathways to maintain intestinal structure and function, (b) feeding the liver with an optimal substrate mix, and (c) providing citrulline and thereby arginine for the whole organism.

Effects of vascular or luminal administration and of simultaneous glucose availability on glutamine utilization by isolated rat small intestine.

This study examined whether the route of glutamine administration and the simultaneous availability of glucose affect intestinal glutamine metabolism. We measured net substrate exchange rates of glutamine and its nitrogenous products in the isolated vascularly and luminally perfused rat small intestine (a) as a function of glutamine provision from either the vascular or the luminal or simultaneously from both sides and (b) as a function of simultaneous availability of glucose from various routes. When glutamine was provided from the lumen, only 19-32% of absorbed glutamine appeared intact in the venous effluent, but the release of metabolic products was 170 +/- 5 nmol N min-1 g-1. This measure of intestinal glutamine metabolism was unchanged when glutamine was available only in the vascular perfusate (164 +/- 6 nmol N min-1 g-1). It increased, however, to 271 +/- 14 nmol N min-1 g-1 (P < 0.001) when glutamine was available simultaneously from both the luminal and the vascular perfusate. Glutamine consumption (-110 +/- 6 vs. -70 +/- 5 or -91 +/- 5 vs. -73 +/- 7 nmol min-1 g-1; P < 0.05 each) and the production of citrulline (11.4 +/- 0.7 vs. 10.0 +/- 0.8 or 9.8 +/- 0.5 vs. 7.8 +/- 0.4 nmol min-1 g-1; P < 0.05 each) or ammonia (124 +/- 7 vs. 88 +/- 4; P < 0.01 or 78 +/- 4 vs. 68 +/- 5 nmol min-1 g-1) decreased when glucose (vascular or luminal perfusate) became available in addition to glutamine. We conclude that glutamine is utilized by the small intestine very efficiently regardless of the route of administration being enteral or parenteral. The two routes can be used interchangeably to provide the intestinal mucosa with glutamine. Glucose and glutamine may partially substitute each other, most likely for the purpose as a metabolic fuel.

Analysis of an oligonucleotide N3'-->P5' phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis.

N3'-->P5' phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, "second generation" chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18-mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3'-->P5' phosphoramidate (amidate) linkages on the 5'-end and five amidate linkages ont he 3'-end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3'-->P5' phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase - high performance liquid chromatography (RP-HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.

An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification.

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.

The detection of oligonucleotide N3'-->P5' phosphoramidate/RNA duplexes with capillary gel electrophoresis.

Oligonucleotide N3'-->P5' phosphoramidates (3'-phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single-stranded RNA fragments utilizing 3'-phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3'-Phosphoramidate 9-mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3'-phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3'-phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation.

Assessment of high-affinity hybridization, RNase H cleavage, and covalent linkage in translation arrest by antisense oligonucleotides.

Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3' P5' phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.

Investigation of the 'n-1' impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase beta-cyanoethyl phosphoramidite method using stepwise sulfurization.

Electrospray ionization mass spectrometry (ESI-MS) of reversed-phase HPLC-purified phosphorothioate oligodeoxynucleotides (S-ODNs), and the single-('n - 1') and double-nucleotide deletion ('n - 2') impurities subsequently isolated from them by preparative polyacrylamide gel electrophoresis (PAGE), has provided direct analytical data for the identification of both S-ODN products and their major oligomeric impurities. The 'n - 1' impurity seen by PAGE consists of a mixture of all possible single deletion sequences relative to the parent S-ODN (n-mer) and results from repetitive, though minor, imperfections in the synthesis cycle, such as incomplete detritylation, or incomplete coupling followed by incomplete capping or incomplete sulfurization. Therefore each possible 'n - 1', 'n - 2', and other short-mer sequence is present only in very low abundance. The conversion of the gel-isolated 'n - 1' impurity from phosphorothioate to phosphodiester followed by base composition-dependent anion-exchange chromatography allowed for independent confirmation of its heterogeneity and quantitation of its various components. ESI-MS of both S-ODN products and their gel-isolated impurities allowed for this first molecular identification of 'n - 1', 'n - 2' and other oligomeric impurities in S-ODNs obtained from state-of-the-art solid-phase synthesis and reversed-phase HPLC purification methods.

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

Lactulose or paromomycin do not affect ammonia generation in the isolated perfused rat small intestine.

It has been hypothesized that the beneficial effect on hepatic encephalopathy of lactulose or neomycin might be exerted by their effect on intermediary glutamine metabolism and ammonia generation within enterocytes. We examined glutamine consumption and the production of alanine and ammonia (net substrate exchange in nmol min-1 g-1) in isolated vascularly and luminally perfused small intestine from rats with and without pretreatment with lactulose (2.0 g/kg) or paromomycin (60 mg/kg). Without pretreatment, 50 mM lactulose or 1 mM paromomycin were equally ineffective to significantly reduce the consumption of arterial glutamine (-92 +/- 5 vs. -80 +/- 6 vs. -71 +/- 6 for controls, lactulose, or paromomycin; mean +/- SEM, n = 6 each, n.s. by analysis of variance), and the production of alanine (41 +/- 3 vs. 44 +/- 3 vs. 61 +/- 7, n.s.) or ammonia (42 +/- 6 vs. 42 +/- 6 vs. 38 +/- 6, n.s.). Similarly, glutamine utilisation, and the release of alanine and ammonia were not different after pretreatment for 10 days. Also, both agents did not reduce glutamine absorption from the lumen (-170 +/- 9 vs. -171 +/- 6 vs. -219 +/- 25, n = 5 each) or the concomitant vascular release of metabolic products alanine (92 +/- 7 vs. 78 +/- 10 vs. 77 +/- 10 vs. 77 +/- 7, n.s.) and ammonia (73 +/- 6 vs. 69 +/- 7 vs. 65 +/- 8, n.s.). Our results do not support the hypothesis, that lactulose or paromomycin reduce ammonia generation by small intestinal mucosa through a specific effect on intermediary glutamine metabolism.