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.

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.

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.

Dipeptide metabolism in the isolated perfused rat small intestine.

The purpose of the present study was to investigate the utilisation of vascularly administered leucyl-leucine (Leu-Leu), alanyl-glutamine (Ala-Gln) and glycyl-glutamine (Gly-Gln) by the isolated vascularly perfused rat small intestine. Fractional extraction rates were 49%, 35.5%, and 12% for Leu-Leu, Ala-Gln, and Gly-Gln (0.15mM) corresponding to a net uptake of -63.5, -31.5, and -17 nmol/min/g wet weight. Nitrogen metabolism in terms of glutamine uptake and release of alanine and ammonia was not different when perfusion with dipeptides or with free amino-acids were compared. No soluble dipeptidase activity was released into the plasma- and cell-free synthetic vascular perfusate. No dipeptide could be recovered from the luminal perfusate. Considering the high fractional extraction rate for Leu-Leu, it is conceivable that dipeptide assimilation may occur also in extramucosal gut tissue. Although dipeptide transport cannot be excluded, dipeptide assimilation in small intestine may involve membrane bound peptidase(s), as in liver.

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.

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.

[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.

Separation of muscarinic acetylcholine receptor entities from rat cerebral cortex by ion exchange chromatography.

Digitonin solubilized muscarinic acetylcholine receptor (mAChR) of rat cerebral cortex membranes were chromatographed on the FPLC anion exchanger Mono Q. [3H]QNB (quinuclidinyl benzilate) and [3H]PZ (pirenzepine) binding activity was retarded from a NaCl free elution buffer and thereby separated from a part of the accompanying proteins. Elution of the column with a continuously increasing NaCl concentration desorbed the radioligand binding activities forming several peaks, two of which were nearly completely separated. Our data show that the mAChR in rat cerebral cortex consists of several entities with different electrical charges.

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.

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.

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.

Inter-organ communication between intestine and liver in vivo and in vitro.

The maintenance of body homeostasis requires a finely tuned system of interorgan communication. The intimate metabolic interrelation between intestine and liver is characterized by the unique anatomic position of both tissues using the portal vein as a private channel with the pancreas in optimal position to modulate hepatic metabolism. Gut-derived peptides (such as glucagon-like peptide-1) appear to be involved in the process of liver regeneration by regulating the release of pancreatic hormones (e.g. insulin). Extensive bowel resection or functional exclusion of small intestine may lead to severe liver dysfunction and even cirrhosis, which may be due to the lack of some intestine-derived and as yet unknown factor(s). Here a close cooperation between small intestinal mucosa and hepatocytes is demonstrated leading to the concept of a metabolic gut-liver unit. This metabolic interaction forms a wide spectrum ranging from the secretion of peptide hormones to changes in (portal-venous) substrate availability or hepatocyte cell volume. Further investigation and identification of the mechanisms of such regulatory processes may be facilitated by combined perfusion of isolated rat intestine and liver. Using this in vitro approach we could demonstrate the presence of metabolic interorgan communication between isolated perfused tissues independent of plasma borne hormones or extrinsic neural control.

[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.

Use of an artificial oxygen carrier in isolated rat liver perfusion: first demonstration of net glucose uptake at physiological portal glucose concentrations using a hemoglobin-free perfusate.

A defect in isolated perfused rat-liver (IPRL) preparations has been proposed to explain discrepancies between in vivo and in vitro findings regarding hepatic glucose metabolism. The aim of the present study was to investigate whether a preparation of IPRL using a synthetic hemoglobin-free perfusate was capable of net glucose uptake and glycogen deposition at physiological portal substrate concentrations. Livers from fed anaesthetized rats were perfused in a recirculating system using a fluorocarbon emulsion as artificial oxygen carrier. Depending on the prevailing glucose concentration, livers exhibited net glucose uptake or release with a threshold value of 5.5-6.0 mM glucose. Net glucose uptake was associated with net glycogen deposition (+0.23 to +0.59 mumol C6 min-1 g-1). From 5.8 mM (n = 3) and 10.0 mM (n = 8), initial concentration glucose levels fell to 5.3 +/- 0.2 mM after 210 min (n = 3) and 6.3 +/- 0.9 mM after 120 min (n = 8), respectively. This was equivalent to a net glucose uptake of -0.16 and -0.45 mumol min-1 g-1. Anoxia reversibly switched hepatic glucose balance from net uptake (-0.42 mumol min-1 g-1) to release (+0.69 mumol min-1 g-1) followed by net uptake (-0.50 mumol min-1 g-1) after reinstitution of aerobic conditions. We conclude that the composition of perfusion media might play a pivotal role for studies of glucose metabolism in the isolated perfused rat liver. In our experimental model, using a hemoglobin-free synthetic medium, net glucose uptake was readily demonstrated at physiological portal substrate concentrations similar to the in vivo situation.

Effects of dexamethasone on glutamine metabolism in the isolated vascularly perfused rat small intestine.

Post-stress metabolism is associated with a large glutamine (Gln) efflux from muscle and an increased Gln utilization by the small intestine. Both appear to be modulated by corticosteroids. The present investigation was performed to better characterize the mechanism of corticoid action on Gln metabolism in an isolated preparation of vascularly perfused rat small intestine. In all perfusions, a synthetic perfusate free from blood components was used with only 0.6 mM Gln and 10 mM glucose as substrates. Irrespective of dexamethasone concentrations in the vascular perfusate (none, 0.25 mg l-1, or 2.5 mg l-1, isolated intestines from normal rats exhibited unchanged extraction rates of Gln (-85 +/- 8, -89 +/- 10, and -87 +/- 16 nmol min-1 g-1) and unchanged production rates of alanine (43 +/- 9, 40 +/- 7, and 51 +/- 5 nmol min-1 g-1) and ammonia (49 +/- 15, 45 +/- 13, and 54 +/- 13 nmol min-1 g-1). Similarly, when intestines were vascularly perfused 2 or 9 days after dexamethasone injection (0.45 mg kg-1 BW), net Gln uptake also remained unchanged (-88 +/- 16 and -84 +/- 11 nmol min-1 g-1). There was, however, a shift in nitrogenous products of Gln metabolism from ammonia (-31% and -38%) to alanine (+16% and +64%). Thus, the failure of dexamethasone to increase Gln uptake in the isolated rat intestine may indicate that rather than acting directly on the mucosa, dexamethasone could regulate intestinal Gln consumption in vivo by indirect mechanisms possibly involving extramucosal tissues. Dexamethasone pretreatment may modulate the pattern of nitrogenous products in portal venous blood presented to the liver and thus support enhanced nitrogen loss through ureagenesis by metabolic cooperation between gut and liver.

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.

Nitrogen absorption from isonitrogenous solutions of L-leucyl-L-leucine and L-leucine: a study in the isolated perfused rat small intestine.

1. We assessed the efficacy of nitrogen absorption from luminal L-leucine (1.2 and 12 mmol/l) and from isonitrogenous L-leucyl-L-leucine (0.6 and 6.0 mmol/l) in a preparation of vascularly and luminally perfused rat small intestine by measuring luminal leucyl-leucine disappearance and venous leucine appearance. 2. No intact dipeptide was found in the vascular perfusate. Leucine-nitrogen absorption, as judged by venous leucine appearance, was as efficient from free leucine (29 +/- 3 and 245 +/- 19 ng-atom min-1 g-1) as from leucyl-leucine (27 +/- 4 and 211 +/- 58 ng-atom min-1 g-1). It was not reduced in the presence of glycyl-L-proline or of the brush-border dipeptidase inhibitors alanine-beta-naphthylamide and cilastatin. 3. After one passage of the whole small intestine, only trace amounts of dipeptide, but large amounts of free leucine, were detected in the luminal effluent. Peptidase activity in the luminal effluent was demonstrated in 100,000 g supernatant and was inhibited by p-hydroxy-mercuribenzoate, but not by brush-border dipeptidase inhibitors. 4. We propose that nitrogen absorption from luminal leucyl-leucine may proceed predominantly via intraluminal peptide hydrolysis and subsequent transport of free leucine. Nevertheless, our findings and conclusions are at variance with previous observations and current opinion on intestinal handling of dipeptides, which may be due, in part, to the different methodological approach.

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.