Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging.

In this paper a novel approach to frequency-domain fluorescence lifetime imaging (FLIM) is described. In a CCD camera a single pixel is defined by a charge pattern on a group of electrodes. By modulation of the pattern of voltages defining the pixel structure it is possible to modulate the sensitivity of the CCD at radio frequency. The modulation enhances the noise performance of the CCD, in contrast to the deterioration in performance seen when an intensifier stage is similarly modulated. The new technology has potential applications to a wide range of assays as well as in conventional FLIM applications. Unlike intensifier-based systems, the directly modulated CCD is physically small, inexpensive, robust and offers superior resolution and noise performance.

Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera.

CCD cameras coupled optically to gated image intensifiers have been used for fast time-resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the 'charge drain' function on an interline-transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.

In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during.

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.

Fractures in children treated with radiotherapy for soft tissue sarcoma.

There is a clear association between multimodal therapy for bone tumors and the development of skeletal complications; however, this has not been addressed in children with soft tissue sarcomas. We reviewed records of the 70 children treated for soft tissue sarcoma of the lower extremity at St. Jude Children's Research Hospital between 1962 and 1991. Of the 12 patients who received radiation after surgical excision of their tumors, three subsequently developed fractures. Two of the three had also received chemotherapy. Our findings indicate that, although the risk of fracture after therapy for soft tissue sarcoma may be multifactorial, radiation may play a significant role. Minimizing the size of surgical incisions, improving radiotherapy techniques, maximizing chemotherapy, and emphasizing physical therapy and appropriate follow up can all serve to decrease long-term toxicities. Such optimal use of therapy could subsequently reduce side effects, such as osteoporosis and muscle and bone atrophy, that predispose patients to fractures.

A longitudinal study of granulocyte colony-stimulating factor levels and neutrophil counts in newborn infants.

PURPOSE: The goal of this study was to longitudinally measure endogenous granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in newborn infants and to attempt to correlate these levels with neutrophil counts. PATIENTS AND METHODS: Samples for complete blood count, G-CSF, and GM-CSF were obtained from groups of healthy full-term infants at 0 (cord blood or nursery admission), 12, 24, 48, and 72 h. Samples were also obtained from premature infants at the above times and at 1 week. G-CSF and GM-CSF levels were measured using bioassays. RESULTS: Levels of G-CSF ranged from < 5 to 53,800 pg/ml. Levels were significantly higher (p < 0.001) in premature infants and decreased over time in all infants. White blood cell counts also decreased over the first week of life. All GM-CSF levels were below the detectable range. CONCLUSIONS: Levels of G-CSF at birth are higher than those seen in adults. These increased levels may partially explain the leukocytosis seen in the first week of life. This, as has been shown in preliminary studies, suggests that infants are capable of an increase in neutrophil count after administration of exogenous G-CSF.

Effectiveness of paclitaxel in treating papillary serous carcinoma of the peritoneum in an adolescent.

A 15-year-old girl with papillary serous carcinoma of the peritoneum was seen with abdominal distention, ascites, and elevated CA 125 tumor marker. After other therapy failed, she achieved complete and continuing remission with paclitaxel.

A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents.

Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and alpha-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 microliters of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 microliters of this cell suspension were incubated with mepacrine alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 microliters of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.

Effect of hematopoietic growth factors on MR images of bone marrow in children undergoing chemotherapy.

PURPOSE: To evaluate the effects of hematopoietic growth factors on magnetic resonance (MR) image signal intensity (SI) of bone marrow in children undergoing neoadjuvant chemotherapy for musculoskeletal malignancies. MATERIALS AND METHODS: Blinded evaluations were performed of T1-weighted and short inversion time inversion recovery images of the pelvis and/or lower extremities of 15 patients undergoing chemotherapy, with or without granulocyte or granulocyte-macrophage colony-stimulating factor. RESULTS: Four of the 11 patients who received growth factors initially had hematopoietic marrow SI that did not change during therapy. All seven with initially fatty marrow had SI changes consistent with reconversion to hematopoietic marrow that coincided temporally with increases in absolute neutrophil counts from minimums of 0-2,580/mm3 to maximums of 10,858-45,880/mm3. The four patients who did not receive growth factors had no MR imaging changes in initially hematopoietic (n = 1) or fatty (n = 3) marrow. CONCLUSION: Hematopoietic activity induced by growth factors can produce changes in bone marrow SI that may simulate bone marrow involvement by musculoskeletal tumors.