Comparison between 1.5T and 3.0T MRI: both field strengths sensitively detect subclinical inflammation of hand and forefoot in patients with arthralgia.

OBJECTIVE: Magnetic resonance imaging (MRI) of small joints sensitively detects inflammation. This inflammation, and tenosynovitis in particular, has been shown to predict rheumatoid arthritis (RA) development in arthralgia patients. These data have predominantly been acquired on 1.0-1.5 T MRI. However, 3.0 T is now commonly used in practice. Evidence on the comparability of these field strengths is scarce and has never included subtle inflammation in arthralgia patients or tenosynovitis. Therefore, we assessed the comparability of 1.5 T and 3.0 T in detecting subclinical inflammation in arthralgia patients. METHOD: A total of 2968 locations (joints, bones, tendon sheaths) in the hands and forefeet of 28 patients with small-joint arthralgia, at risk for RA, were imaged on both 1.5 and 3.0 T MRI. Two blinded readers independently scored erosions, osteitis, synovitis, and tenosynovitis, in line with the Rheumatoid Arthritis Magnetic Resonance Imaging Score (RAMRIS). Features were summed into inflammation (osteitis, synovitis, tenosynovitis) and RAMRIS (inflammation and erosions). Agreement was assessed with intraclass correlation coefficients (ICCs) for continuous scores and after dichotomization into presence or absence of inflammation, on patient and location levels. RESULTS: Interreader ICCs were excellent (> 0.90). Comparing 1.5 and 3.0 T revealed an ICC of 0.90 for inflammation and RAMRIS. ICCs for individual inflammation features were: tenosynovitis 0.87 (95% confidence interval 0.74-0.94), synovitis 0.65 (0.24-0.84), and osteitis 0.96 (0.91-0.98). Agreement was 83% for inflammation and 89% for RAMRIS. Analyses on the location level showed similar results. CONCLUSION: Agreement on subclinical inflammation between 1.5 T and 3.0 T was excellent. Although synovitis scores were slightly different, synovitis often occurs simultaneously with other inflammatory signs, suggesting that scientific results on the predictive value of MRI-detected inflammation for RA, obtained on 1.5 T MRI, can be generalized to 3.0 T MRI.

Do magnetic resonance imaging-detected erosions predict progression to rheumatoid arthritis in patients presenting with clinically suspect arthralgia? A longitudinal study.

Objective: Radiographic joint erosions are a hallmark of rheumatoid arthritis (RA). Magnetic resonance imaging (MRI) is more sensitive than radiographs in detecting erosions. It is unknown whether MRI-detected erosions are predictive for RA development in patients with clinically suspect arthralgia (CSA). Therefore, we investigated the prognostic value of MRI-detected erosions, defined as any MRI erosion, or MRI erosion characteristics that were recently identified as specific for RA in patients with evident arthritis. Method: Patients presenting with CSA (n = 490) underwent contrast-enhanced 1.5 T MRI of the wrist, metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints. MRIs were scored according to the Rheumatoid Arthritis Magnetic Resonance Imaging Scoring system (RAMRIS). Presence of any MRI erosion (present in < 5% of symptom-free controls) and RA-specific erosion characteristics as identified previously (grade ≥ 2 erosions, erosions in MTP5, erosions in MTP1 if aged < 40 years) were studied with clinically apparent inflammatory arthritis development as outcome. Analyses were corrected for age and MRI-detected subclinical inflammation. Results: Erosions were present in 20%. Presence of any MRI erosion was not associated with arthritis development [multivariable analysis hazard ratio (HR) 0.97 (95% confidence interval 0.59-1.59)]. The different RA-specific erosion characteristics were not predictive [grade ≥ 2 HR 1.05 (0.33-3.34), erosions in MTP5 HR 1.08 (0.47-2.48), and MTP1 if aged < 40 years HR 1.11 (0.26-4.70)]. Erosion scores were higher in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative patients (median 2.0 vs 1.0, p = 0.002), and related to more subclinical inflammation. Within both subgroups, MRI erosions were not predictive. Conclusions: MRI-detected erosions in hands and feet were not predictive for inflammatory arthritis development. Therefore, evaluating MRI for erosions in addition to subclinical inflammation does not provide added clinical value in CSA.

Pneumotoxicosis in sheep caused by ingestion of trema micrantha.

Trema micrantha, a fast-growing tree distributed throughout the Americas, produces palatable leaves that have been associated with hepatic necrosis and acute death when consumed by livestock. This report describes fatal pulmonary disease of sheep triggered by consumption of Trema micrantha. Affected sheep had severe progressive dyspnea for a few days before death. Subcutaneous and mediastinal emphysema, reddened lungs, interalveolar septal thickening, and diffuse type II pneumocyte proliferation were the main pathological findings. After ingesting 77.5 and 102.5 g/kg (divided in 3 doses, at 30-day intervals) of T. micrantha leaves, 2 additional sheep developed the same condition. These findings indicate that T. micrantha toxicosis should be considered in the differential diagnosis of ovine respiratory disease.

Mutant SOD1 detoxification mechanisms in intact single cells.

Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cell-by-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS.

Imaging biochemistry inside cells.

Proteins provide the building blocks for multicomponent molecular units, or pathways, from which higher cellular functions emerge. These units consist of either assemblies of physically interacting proteins or dispersed biochemical activities connected by rapidly diffusing second messengers, metabolic intermediates, ions or other proteins. It will probably remain within the realm of genetics to identify the ensemble of proteins that constitute these functional units and to establish the first-order connectivity. The dynamics of interactions within these protein machines can be assessed in living cells by the application of fluorescence spectroscopy on a microscopic level, using fluorescent proteins that are introduced within these functional units. Fluorescence is sensitive, specific and non-invasive, and the spectroscopic properties of a fluorescent probe can be analysed to obtain information on its molecular environment. The development and use of sensors based on the genetically encoded variants of green-fluorescent proteins has facilitated the observation of 'live' biochemistry on a microscopic level, with the advantage of preserving the cellular context of biochemical connectivity, compartmentalization and spatial organization. Protein activities and interactions can be imaged and localized within a single cell, allowing correlation with phenomena such as the cell cycle, migration and morphogenesis.

Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane.

Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)-tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime imaging microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.

Imaging FRET between spectrally similar GFP molecules in single cells.

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.

Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

The immunohistochemical localization of the non-specific lipid transfer protein (sterol carrier protein-2) in rat small intestine enterocytes.

A 13 kDa protein was isolated from rabbit small intestine brush-border membrane vesicles that was postulated to be involved in intestinal phosphatidylcholine (PC) and cholesterol uptake. This protein has cholesterol and PC-transfer activity in vitro (Turnhofer, H. et al. (1991) Biochim. Biophys. Acta 1064, 275-286) and has a molecular mass and isoelectric point similar to that of the non-specific lipid transfer protein (nsL-TP, identical to sterol carrier protein-2). In addition, the first 28 N-terminal amino acid residues of the 13 kDa protein are nearly identical to nsL-TP from different species (Lipka, G. et al. (1995) J. Biol. Chem. 270, 5917-5925). In view of its possible role in intestinal lipid absorption, the localization of nsL-TP in rat small intestine was investigated using immunohistochemistry and immunoblotting. It is shown that nsLTP is predominantly localized in a subapical zone of the enterocyte but not in the brush-border membrane, thereby excluding a role in lipid uptake of this protein at the level of the plasma membrane. nsL-TP co-localized with the peroxisomal marker PMP70, underscoring earlier observations that nsL-TP is a peroxisomal protein. nsL-TP was found to be present along the entire length of the small intestine. The 58 kDa cross-reactive protein that was recently identified as a peroxisomal thiolase was shown to be present only in a small segment approximately halfway down the jejunum. The close apposition of the peroxisomes with the apical membrane and the discrete distribution of the 58 kDa protein may indicate that these organelles play a role in the intracellular processing of absorbed lipids.

All-solid-state lock-in imaging for wide-field fluorescence lifetime sensing.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique that is increasingly being used in the life sciences during the past decades. However, a broader application of FLIM requires more cost-effective and user-friendly solutions. We demonstrate the use of a simple CCD/CMOS lock-in imager for fluorescence lifetime detection. The SwissRanger SR-2 time-of-flight detector, originally developed for 3D vision, embeds all the functionalities required for FLIM in a compact system. The further development of this technology and its combination with light-emitting-and laser diodes could drive a wider spreading of thuse of FLIM including high-throughput applications.

Measurement of gingival swelling from dental casts by generation of a moiré pattern with laser light.

The aim of the study was to develop a sensitive measuring method enabling direct evaluation of gingival swelling to be made as registered on dental casts. On two separate occasions, when different degrees of severity of gingival inflammation were present in the same subject, reversible hydrocolloid impressions were taken of the mandible. The casts obtained were located successively in identical three-dimensional relationships in a field of interference fringes generated by two intersecting beams of collimated helium-neon laser light, and were photographed. The evaluation of the moiré pattern obtained directly by superimposition of the two images of the surface studied indicated that a decrease in gingival height of 0.38 mm in the direction of the camera had occurred between the two occasions. The use of a computer-based image-processing system considerably improved the visibility of the pattern. The reproducibility of the impression technique, as well as the relocation and superimposing techniques, proved satisfactory at the moiré resolution used (0.19 mm). The method has potential application in clinical experimental research, and therefore warrants further evaluation.

The effect of systemic metronidazole after non-surgical treatment in moderate and advanced periodontitis in young adults.

The effect of adjunctive systemic metronidazole was studied in patients with moderate and advanced periodontitis recalcitrant to comprehensive non-surgical treatment. The material originated from a randomly selected part of the population aged 31 to 40 years. After non-surgical treatment of 149 patients, 98 with persisting pathological pockets greater than or equal to 5 mm (52 men and 46 women) became the subjects for the study. Clinical parameters were registered and pocket contents subjected to laboratory analysis. The subjects were randomized into two groups according to a code list known only by the manufacturer and the statistician. The test group took three 400 mg metronidazole tablets daily for 1 week and the control group took placebo tablets. Reassessment 6 months later showed statistically significant clinical improvement, with a reduction in the number of sites greater than or equal to 5 mm in both test and control groups. Complete healing, with no pockets greater than or equal to 5 mm, was noted in 30% of the test group and 9% of the control group. The difference is statistically significant and shows the supplementary effect of adjunctive metronidazole in non-surgical treatment of moderate and advanced periodontitis.

The approximal bone height and intrabony defects in young adults, related to the salivary buffering capacity and counts of Streptococcus mutans and Lactobacilli.

Using a computerized technique the bone height and prevalence of approximal periodontal intrabony defects were assessed on posterior bite-wing radiographs from 151 young adults. The results were related to the buffering capacity and counts of Streptococcus mutans and lactobacilli in whole stimulated saliva. The mean distance from the cement-enamel junction to the alveolar bone crest was greater in the high buffering group than in the low buffering group (p less than 0.05), and particularly in non-smokers (p less than 0.01). Intrabony defects were more common in the low buffering group (p less than 0.05) and in women (p less than 0.001).

The relation between tooth eruption and alveolar crest height in a human skeletal sample.

It is commonly assumed that alveolar crest height increases with continuing tooth eruption unless affected by marginal inflammation. To test this hypothesis, the relation between eruption and alveolar crest height was examined in skulls from a sample consisting of the remains of 244 individuals from the late medieval period. The mandibular first and second molars and second premolars were analysed. The age of the skulls was determined on the basis of dental development and molar attrition. Radiographs were taken and points representing the levels of the inferior dental canal (IDC), root apices (AP), alveolar crest (AC), cementum-enamel junction (CEJ) and occlusal surface were determined on the radiographs. The level of the IDC was used as a reference not changing with age. The distances between the points were measured with a help of a computer-digitizer system. Variable IDC-AP increased with age, indicating continuous eruption of the teeth. The distance between AC and CEJ also increased while the distance between IDC and AC remained constant, showing that the alveolar crest height did not increase accordingly. The lack of inflammatory changes on the alveolar bone surface suggests that occlusal attrition may be compensated for by continuous eruption without bone growth in the alveolar margin.

EPIX: an interactive computer application program for cross-sectional epidemiological, periodontal investigations from X-ray films.

This article introduces the interactive computer application program EPIX and the associated personal computer/digitizer system. The system is used to measure marginal alveolar bone height and to record angular alveolar bone defects, defective margins of dental restorations and dental calculus from 5 X-magnified periapical radiographs in cross-sectional epidemiological investigations. The 134-kBytes compiled BASIC program provides 17 menu options. A complete recording of interproximal tooth surfaces takes on average 20 minutes per subject. Data from 200 subjects can be stored on one 5.25" floppy disk. EPIX transforms data files into text files to enable transfer to main-frame computer for statistical analyses. Data displays on VDU or paper are available. Routines for data location and processing, file processing and visual display of an operator's manual provide additional facilities. EPIX has been used to process an epidemiological material of 912 subjects and has proved to be useful.

Quality of intraoral radiographs sent by private dental practitioners for therapy evaluation by the Social Insurance Office.

Nearly all Swedish private dentists are affiliated with the Dental Care Insurance. Proposed treatment plans, including radiographs, are submitted to the Social Insurance Office for prior approval. In this study the quality of a sample of intraoral radiographs submitted by 404 private dental practitioners was analysed. Forty-four per cent of 3,708 periapical radiographs, simultaneously evaluated by two examiners, were satisfactory. The most frequent errors were excessive vertical angulation (18 per cent), missing apex (12 per cent), film positioning error (11 per cent) and underexposure (10 per cent). Fifty-five per cent of 294 bitewing radiographs were satisfactory; the major error was missing bone margin (33 per cent). The radiographs of 15 per cent of the 2,844 teeth proposed for therapy had insufficient radiographic documentation, six per cent because of missing radiograph and nine per cent because of missing apex on the radiograph. These errors applied to every second dentist. Compared to a similar study ten years ago, the present results do not indicate that the quality of intraoral radiographs has improved.

Undifferentiated carcinoma of the pancreas in a cockatiel (Nymphicus hollandicus): case report / Carcinoma indiferenciado de pâncreas em calopsitas (Nymphicus hollandicus): relato de caso

Undifferentiated carcinoma of the pancreas is a malignant neoplasm that is uncommon among domestic species, especially cockatiels (Nymphicus hollandicus), one of the most popular birds kept as a pet throughout the world. The aim of this study was to describe the occurrence of an undifferentiated carcinoma in the pancreas of a cockatiel. A bird, an adult male that died naturally with swelling in the abdominal region, was referred to necropsy. Macroscopic examination showed poor body condition, the coelomic cavity filled with liquid and a white mass attached to the pancreas and other smaller masses attached to the duodenum. Tissue samples and organs were harvested and fixed in 10% buffered formalin, then routinely processed for histopathology and stained with hematoxylin and eosin. Microscopic analysis demonstrated an epithelial neoplasia with a predominantly solid pattern, lymphatic invasion and involvement of the intestinal serous membrane. These findings indicate the occurrence of an undifferentiated pancreatic carcinoma in a cockatiel that was diagnosed by histopathology.(AU)

O carcinoma indiferenciado de pâncreas é uma neoplasia maligna, incomum entre as espécies domésticas, especialmente em calopsitas (Nymphicus hollandicus), uma das aves mais populares como animal de companhia no mundo. O objetivo deste trabalho foi descrever a ocorrência de carcinoma indiferenciado de pâncreas em Nymphicus hollandicus. Uma ave, macho adulto, com morte natural e com aumento de volume em região abdominal, foi encaminhada para necropsia. Ao exame macroscópico foram observados mau estado corporal, cavidade celômica repleta de líquido e massa esbranquiçada aderida ao pâncreas e outra menor aderida ao duodeno. Amostras de tecidos e órgãos foram colhidas em formol 10% tamponado, processadas rotineiramente para histopatologia e coradas por hematoxilina e eosina. Na microscopia foi observada neoplasia epitelial com padrão predominantemente sólido, com invasão linfática e implantação na serosa intestinal. Com esses achados, comprovou-se a ocorrência de carcinoma indiferenciado de pâncreas em Nymphicus hollandicus, o qual pode ser diagnosticado por meio de histopatologia.(AU)

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30-40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34(+) AML patients. At diagnosis, subfractions of the CD45(dim)CD34(+)CD38(dim/-) compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45(dim)CD34(+) blast compartment in 6 out of 7 patients. Moreover, within CD45(dim)CD34(+)CD38(dim/-) fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.