An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.     

Cerebrospinal fluid acetylcholinesterase activity after long-term treatment with donepezil and rivastigmina

At present acetylcholinesterase (AChE) inhibitors (AChEIs) represent the only reliable therapeutic resource for symptomatic treatment of Alzheimer Disease (AD). This study was designed to assess the effects of 6-12 month treatment with AChEIs donepezil and rivastigmine on cerebrospinal fluid (CSF) AChE and butyrylcholinesterase (BuChE) activity in AD patients. The pattern of AChE isoforms (G4, G1, G2) before and after treatment was investigated as well. In AD patients treated with donepezil a significant increase of CFS AChE activity was observed, whereas treatment with rivastigmine induced a significant decrease of AChE activity. Both drugs did not change BuChE activity and tended to restore the physiological pattern of AChE isoform. The possible significance of the influence of AChEIs on CSF AChE activity and isoforms is discussed.     

Neuromagnetic somatosensory homunculus: a non-invasive approach in humans

The somatosensory homunculus has been identified during stimulation of median (at wrist and elbow), femoral, tibial and pudendal nerves of the left hemibody via the neuromagnetic imaging technique. The somatic representations of different body districts have been localized in the somatosensory cortex, by means of an equivalent dipole localization algorhythm. Dipole locations agree with the well-known somatotopic organization obtained with invasive techniques. The proposed method is, therefore, an important investigating tool for studies on normal and diseased subjects.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-microl volume of urine is added to a carbonate-hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70 degrees C for 10 min. The 'dabsylated' product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C18 column (250 x 4.6 mm, 5 microm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5-40.8 mg/m3), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Critical review of 215 cases of non-palpable mammary lesions]

PURPOSE: The natural history of human breast cancer shows that lesion size correlates directly with nodal metastases and distant spread. Nodal metastases are found in only 6% of cases in the preclinical stage of the tumor and therefore imaging must detect a breast cancer before it becomes palpable. We reviewed 215 nonpalpable breast lesions studied in the last 10 years to assess observers performance and ultimately improve the interpretation of suspicious mammograms, evaluating "cost" in terms of the ratio between benign and malignant lesions (B/M). MATERIAL AND METHODS: From 1988 to October 1998, two hundred and fifteen women with nonpalpable breast lesions suspected at mammography were examined. The lesions were removed after stereotaxic or US location and a radiograph of the surgical specimen was always performed. Mammographic patterns were interpreted retrospectively by two blinded radiologist experienced in breast imaging and specialized in locating nonpalpable breast lesions. Mammographic patterns were classified as poorly/highly suspicious calcifications, regular/irregular masses, spiculated masses, masses with calcifications and parenchymal distortions. Radiographic findings were compared with surgical results and the data used to calculate the B/M, positive predictive value (PPV) for malignancy and the trend of operator's performance. RESULTS: Modern techniques permit to detect a very high number of in situ breast carcinomas. Nineteen of 22 lesions (86%) were detected by mammography as highly suspicious calcifications, 2/22 as spiculated masses and 1/22 as a mass with calcifications. No in situ carcinoma was detected as an irregular mass. All regular masses were proven to be benign at histology. B/M analysis showed a decreasing trend (from 1.94 in the first 3 years to .57 in 1994-96, to .83 in 1997-98) and an overall value of .90. The PPV for malignancy was 83.33% for spiculated masses, 65.5% for highly suspicious calcifications, 63.63% for irregular masses, 47.05% for masses with more or less dysmorphic calcifications, 32.65% for poorly suspicious calcifications, 8.33% for parenchymal distortions and 0% for regular masses. DISCUSSION AND CONCLUSIONS: All spiculated masses and highly suspicious calcifications and microcalcifications should be removed. Biopsy is recommended in parenchymal distortions, despite its low predictive value for malignancy, because these lesions are uncommon and the cost of biopsy is therefore acceptable. Needle aspiration or long-term monitoring can be reconsidered for irregular masses and poorly suspicious microcalcifications. Finally, relative to possible different interpretations of mammographic patterns by center and operator's experience, we suggest that the PPV for every single pattern be continually reassessed based on personal case records rather than on literature data. This holds true especially for microcalcifications.     

Functional localization of the sensory hand area with respect to the motor central gyrus knob

The aim of this work was to investigate the topography of the primary sensory hand cortex with magnetoencephalography in order to define the functional anatomy of this area in healthy humans. Previous studies denoted an inverted Ohm or an horizontal epsilon shaped knob on the pre-central gyrus as a landmark for the motor hand area; therefore a systematic difference between the orientation of the source for thumb with respect to little finger should be observed. We found this systematic difference, but the direction of the sources activated during thumb and little finger stimulation did not converge, as would be expected if only the Ohm convexity is activated: in fact our results suggest that thumb sensory area also extends to the area lateral to this convexity.     

Spin-echo anatomy of the larynx with magnetic resonance at 1.5 T

The larynges of 8 healthy and informed volunteers were studied with a superconductive MR unit at 1.5 T together with those of 10 patients with extralaryngeal pathologic conditions. The study was performed with round surface coils (5") and with dedicated sellar coils in the anterior neck. Slices were 5 mm thick, and acquired on the coronal, axial, and sagittal planes, with T1-weighting; axial scans were repeated in the same locations with double echoes, with proton-density and T2-weighting. Five patients underwent additional scans after Gd-DTPA. The larynx of a semi-frozen cadaver was examined with sellar surface coils, on similar scanning planes and with similar pulse sequences to those described above; the larynx was removed, investigated with mammographic technique, and subsequently analyzed with thin CT slices and a high-resolution reconstruction algorithm for the study of laryngeal cartilage. Axial anatomical sections were then compared with MR and CT scans, and the anatomical structures were recognized on the triplanar MR scans of a volunteer's larynx. Besides MR anatomy of supporting laryngeal structures, the authors describe in detail the muscles, plicae, spaces and cavities which can be identified on the various planes, together with the changes in signal after Gd-DTPA.