Fluorescence polarization as an early measure of T-lymphocyte stimulation.
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Cell Separation↗
Biomedical subjects
M Deutsch
Publications and source records attributed to M Deutsch.
Determination of cellular thiol levels in individual viable lymphocytes by means of fluorescence intensity and polarization.
Cellular thiol levels regulate lymphocyte proliferation and death and play a significant role in the immune response. Therefore, the ability to analyze the total protein and non-protein thiol compounds and their distribution among individual living lymphocytes is of great importance. A quantitative measurement of intracellular sulphydryl groups in living lymphocytes using the Cellscan mark F (CS-F) cytometer, in conjunction with the probe CMFDA, is described. This technique permits the detection, identification, and study of sub-populations and single cells in a sample of heterogeneous lymphocytes. The Cellscan apparatus is a laser based scanning cytometer incorporating a unique cell carrier which allows repeated, high-precision measurements of fluorescence intensity (FI) and fluorescence polarization (FP) to be made on intact individual living cells under controlled physiological conditions. The discernible effect of fluorophore molecules bound to thiols having a higher FP than free molecules was used to estimate their relative fractions in living lymphocytes. The results were more conspicuous when the ratio between FP measured at two wavelengths (FPR) of the fluorogenic molecules was used for analysis. In addition, the intracellular dynamic changes in the FI, FP and FPR of the fluorescent probe were also monitored. The cellular sulphydryl content of each lymphocyte within a population was recorded by the CS-F, and sub-populations or individual cells were classified according to their thiol levels and their metabolic rates. Changes in thiol concentration were observed following mitogenic activation of peripheral lymphocytes.
Ethylmaleimide↗
The trace and subgrouping of lymphocyte activation by dynamic fluorescence intensity and polarization measurements.
Cell activation involves conformational changes of cytosolic enzymes, and/or their regulatory proteins, as well as intracellular matrix re-organization. In this work, these changes were monitored by dynamic measurements of fluorescence polarization in single cells incubated with or without phytohaemagglutinin (PHA), using the Cellscan mark S (CS-S) cytometer. This instrument and the procedure used proved to be a powerful tool for distinguishing subpopulations of cells. Grouping of cells by their staining rates (the time rate of change of the fluorescence intensity) yielded three major subgroups. For each subgroup, the fluorescence depolarization (FDP) induced by the incubation with PHA was measured. The kinetics of the subgroups indicate that the major FDP is contributed by the cells with the lowest staining rate. This FDP is approximately 1.5 times greater than that of a bulk population. It is believed that the analysis of kinetic probing might yield an important and more sensitive method for functional marking of subgroups of cells by their response characteristics.
Cell Separation↗
Intron-exon structures of eukaryotic model organisms.
To investigate the distribution of intron-exon structures of eukaryotic genes, we have constructed a general exon database comprising all available intron-containing genes and exon databases from 10 eukaryotic model organisms: Homo sapiens, Mus musculus, Gallus gallus, Rattus norvegicus, Arabidopsis thaliana, Zea mays, Schizosaccharomyces pombe, Aspergillus, Caenorhabditis elegans and Drosophila. We purged redundant genes to avoid the possible bias brought about by redundancy in the databases. After discarding those questionable introns that do not contain correct splice sites, the final database contained 17 102 introns, 21 019 exons and 2903 independent or quasi-independent genes. On average, a eukaryotic gene contains 3.7 introns per kb protein coding region. The exon distribution peaks around 30-40 residues and most introns are 40-125 nt long. The variable intron-exon structures of the 10 model organisms reveal two interesting statistical phenomena, which cast light on some previous speculations. (i) Genome size seems to be correlated with total intron length per gene. For example, invertebrate introns are smaller than those of human genes, while yeast introns are shorter than invertebrate introns. However, this correlation is weak, suggesting that other factors besides genome size may also affect intron size. (ii) Introns smaller than 50 nt are significantly less frequent than longer introns, possibly resulting from a minimum intron size requirement for intron splicing.
Animals↗
Results of multifield conformal radiation therapy of nonsmall-cell lung carcinoma using multileaf collimation beams.
A five-field conformal technique with three-dimensional radiation therapy treatment planning (3-DRTP) has been shown to permit better definition of the target volume for lung cancer, while minimizing the normal tissue volume receiving greater than 50% of the target dose. In an initial study to confirm the safety of conventional doses, we used the five-field conformal 3-DRTP technique. We then used the technique in a second study, enhancing the therapeutic index in a series of 42 patients, as well as to evaluate feasibility, survival outcome, and treatment toxicity. Forty-two consecutive patients with nonsmall-cell lung carcinoma (NSCLC) were evaluated during the years 1993-1997. The median age was 60 years (range 34-80). The median radiation therapy (RT) dose to the gross tumor volume was 6,300 cGy (range 5,000-6,840 cGy) delivered over 6 to 6.5 weeks in 180-275 cGy daily fractions, 5 days per week. There were three patients who received a split course treatment of 5,500 cGy in 20 fractions, delivering 275 cGy daily with a 2-week break built into the treatment course after 10 fractions. The stages of disease were II in 2%, IIIA in 40%, IIIB in 42.9%, and recurrent disease in 14.3% of the patients. The mean tumor volume was 324.14 cc (range 88.3-773.7 cc); 57.1% of the patients received combined chemoradiotherapy, while the others were treated with radiation therapy alone. Of the 42 patients, 7 were excluded from the final analysis because of diagnosis of distant metastasis during treatment. Two of the patients had their histology reinterpreted as being other than NSCLC, 2 patients did not complete RT at the time of analysis, and 1 patient voluntarily discontinued treatment because of progressive deterioration. Median follow-up was 11.2 months (range 3-32.5 months). Survival for patients with Stage III disease was 70.2% at 1 year and 51.5% at 2 years, with median survival not yet reached. Local control for the entire series was 23.3+/-11.4% at 2 years. However, for Stage III patients, local control was 50% at 1 year and 30% at 2 years. Patients who received concurrent chemotherapy had significantly improved survival (P = 0.002) and local control (P = 0.004), compared with RT alone. Late esophageal toxicity of > or =Grade 3 occurred in 14.1+/-9.3% of patients (3 of 20) receiving combined chemoradiotherapy, but in none of the 15 patients treated with RT alone. Pulmonary toxicity limited to Grades 1-2 occurred in 6.8% of the patients, and none developed > or =Grade 3 pulmonary toxicity. Patients with locally advanced NSCLC, who commonly have tumor volumes in excess of 200 cc, presenta challenge for adequate dose delivery without significant toxicity. Our five-field conformal 3-DRTP technique, which incorporates treatment planning by dose/volume histogram (DVH) was associated with minimal toxicity and may facilitate dose escalation to the gross tumor.
Adenocarcinoma↗
Reactivity of peripheral blood lymphocytes to oxidized low-density lipoprotein: a novel system to estimate atherosclerosis employing the Cellscan.
BACKGROUND: The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population. HYPOTHESIS: In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls. METHODS: The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases. RESULTS: Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 micrograms/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 micrograms/ml). Maximal depolarization was shown upon triggering with 2 micrograms/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 +/- 0.05 and 1.028 +/- 0.055, respectively). CONCLUSION: These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.
Aged↗
Re-irradiation for recurrent rhabdomyosarcoma of the nasopharynx in a child.
There are few clinical situations where high dose re-irradiation with curative intent is indicated in children. Because of the potential for serious late sequelae, re-irradiation is considered only in the unusual situation where there is just local recurrence and surgery and/or chemotherapy are unlikely to provide long-term control. The authors present a case of nasopharynx rhabdomyosarcoma in a child which recurred after high dose irradiation and chemotherapy and was then re-irradiated to a high dose. The child is well without any serious sequelae more than 13 years following completion of the second course of radiotherapy.
Antineoplastic Agents↗
Enhanced uptake of [3H] spermidine by 9L rat brain tumors after direct intratumoral infusion of inhibitors of enzymes of the polyamine biosynthetic pathway.
We have been exploring the feasibility of delivering ionizing radiation to brain tumor cells by using tritium labeled polyamines. Polyamines are taken up preferentially by dividing cells and form noncovalent bonds with DNA. Their uptake can be enhanced by drugs which deplete endogenous polyamines. To test this in vivo, 9L cells were implanted in the striatal region of the brain in male Fisher 344 rats. Osmotic pumps containing trace amounts of [3H] spermidine or [3H] putrescine with either difluoromethylornithine or combinations of 3 inhibitors of enzymes of the polyamine biosynthetic pathway were implanted subcutaneously and were connected to intratumoral cannulas. After 14-16 days the brains were removed and sliced in the coronal plane. The diameters of the tumors were measured and tumor tissue was dissected from each slice, weighed and lysed for scintillation counting. It was found that difluoromethylornithine enhanced the uptake of [3H] putrescine while a combination of inhibitors of enzymes of the polyamine biosynthetic pathway enhanced the uptake of [3H] putrescine and [3H] spermidine producing a localized region of radioactivity in the 9L tumor. It is estimated that if the [3H] polyamines were at higher specific activity (commercially available), instead of the trace dose given here, the [3H] polyamine uptake would be sufficient to kill 9L tumor cells within a 2 to 3 week period.
Adenosylmethionine Decarboxylase↗
Preparation of a diagnostic antigen of human melanoma based on lymphocyte activation as measured by intracellular fluorescein fluorescence polarization.
The intracellular fluorescein fluorescence polarization (IFFP) test indicates that peripheral blood lymphocytes (PBL) of cancer patients display stimulatory sensitivity to a short incubation with specific tumor protein extracts. In this work, a human lymphocyte activation melanoma antigen (LAMA) was purified from supernatant of a human melanoma cell line (L1M1), which could specifically stimulate lymphocytes of melanoma patients. The results showed a significant stimulation of lymphocytes from healthy donors after incubation with phytohaemagglutinin (PHA), while no stimulation was observed after incubation with LAMA. On the other hand, lymphocytes from melanoma patients showed a significant stimulation with LAMA, while generally showing minor or no stimulation with PHA. Melanoma specificity of LAMA was demonstrated by no response in lymphocytes from patients of lung, colon, or breast cancer. The purified fraction is therefore considered to be a shared tissue-specific antigen which may be useful in immunodiagnosis and immunotherapy of melanoma.
Antigens, Neoplasm↗
Ductal carcinoma in situ recurrent on the chest wall after mastectomy.
Two patients with local recurrence on the chest wall subsequent to mastectomy for ductal carcinoma in situ (DCIS) are presented. One recurrence was invasive carcinoma and the second was DCIS. Excision and chest wall irradiation, together with chemotherapy in the first patient, have provided subsequent disease-free survivals of 6 and 12 years respectively. Although mastectomy for DCIS is almost always curative, the possibility of local recurrence requires careful surveillance.
Adult↗
Pancreatic cancer in a young adult after treatment for Hodgkin's disease.
The authors present a case of adenocarcinoma of the head of the pancreas, which arose 14 years after extended field irradiation for Stage IIA Hodgkin's disease. The patient was aged 37 years at the time of the pancreatic cancer, which was situated within the previously irradiated volume.
Adenocarcinoma↗
Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns.
How exon-intron structures of eukaryotic genes evolved under various evolutionary forces remains unknown. The phases of spliceosomal introns (the placement of introns with respect to reading frame) provide an opportunity to approach this question. When a large number of nuclear introns in protein-coding genes were analyzed, it was found that most introns were of phase 0, which keeps codons intact. We found that the phase distribution of spliceosomal introns is strongly correlated with the sequence conservation of splice signals in exons; the relatively underrepresented phase 2 introns are associated with the lowest conservation, the relatively overrepresented phase 0 introns display the highest conservation, and phase 1 introns are intermediate. Given the detrimental effect of mutations in exon sequences near splice sites as found in molecular experiments, the underrepresentation of phase 2 introns may be the result of deleterious-mutation-driven intron loss, suggesting a possible genetic mechanism for the evolution of intron-exon structures.
Base Sequence↗
Meningioma after radiotherapy for Hodgkin's disease.
The most common second primary tumors after treatment of childhood Hodgkin's disease are leukemia, lymphoma, breast cancer, soft tissue sarcoma, and thyroid cancer. Although intracranial meningioma has been reported after radiotherapy to the scalp for benign conditions and for intracranial primary brain tumors, this appears to be an extremely rare sequelae of treatment for Hodgkin's disease. The authors describe a 15-year-old boy who underwent radiotherapy for Hodgkin's disease and in whom a meningioma developed in the posterior fossa 27 years later.
Adolescent↗
Radiotherapy review on national surgical adjuvant breast and bowel project (NSABP) phase III breast cancer clinical trials: is there a need for submission of portal/simulation films?
In all National Surgical Adjuvant Breast and Bowel Project (NSABP) breast cancer trials in which patients are treated with lumpectomy and postoperative breast irradiation, the quality assurance requirements dictate submission of a completed radiotherapy data form with the stated administered doses, the volumes treated, treatment prescription and daily treatment record sheets, dosimetry and calculation sheets, isodose distributions on the breast contour, photos of the patient in the treatment position, and portal or simulation films. A review of radiotherapy data on 1,982 patients who had lumpectomies accrued to seven recent NSABP breast cancer studies revealed only 2 patients who were judged to have inadequate fields. In both cases, a very small portion of the breast tissue was not included in the irradiated volume as demonstrated by the submitted films. On this basis, it was argued that submission of portal or simulation films for patients receiving postlumpectomy breast irradiation is not necessary. However, there was concern as to the incidence of patients with a possible excess amount of lung tissue included in the irradiated volume. To address this concern, the amount of irradiated lung was determined for the first 208 patients who had lumpectomies, with submitted data entered into the recent NSABP pathologic node-positive protocol B-28. Current NSABP radiation therapy guidelines suggest limiting the thickness of the irradiated lung in the portal beams to < or =3 cm. Only two patients (<1%) were found to have >3 cm of irradiated lung tissue in the treatment volume. Portal film submission is a considerable inconvenience to the individual institutions and is costly in terms of shipping, handling, and storage. These results indicate that submission of portal films is not a necessary part of quality assurance in NSABP breast cancer protocols. The NSABP has therefore eliminated the requirement for routine submission of portal films in protocols for which radiotherapy is not part of the test question.
Breast↗
Influence of radiation on late complications in patients with free fibular flaps for mandibular reconstruction.
Carcinoma of the mandible is a disease that evokes images of devastating functional and cosmetic outcomes. Most of these malignancies require treatment with surgical resection and perioperative irradiation (XRT). To minimize the incidence of postoperative complications, the timing of perioperative XRT has been questioned. This study reviewed 140 patients at M.D. Anderson Cancer Center over a 7-year period who underwent mandibular resection and reconstruction with a free fibular flap. The patients were divided into the following four groups: (1) preoperative XRT followed by immediate reconstruction, (2) preoperative XRT followed by delayed reconstruction, (3) postoperative XRT, and (4) no XRT. The complications studied included exposure of bone and hardware, orocutaneous fistula, osteoradionecrosis, partial and complete flap loss, and severe cervical contractures. Of the 140 patients studied, 59 (42%) had at least one complication. Complications per group were the following: group 1, 45%; group 2, 46%; group 3, 47%; and group 4, 28%. The results show that the incidence of complications is relatively equal between groups that received preoperative vs. postoperative XRT.
Combined Modality Therapy↗
Fulminant hepatic failure as a presenting paraneoplastic manifestation of Hodgkin's disease.
Malignancies may uncommonly present as fulminant hepatic failure and, due to the rarity of such an occurrence, they may easily be overlooked as one of its possible causes. An unusual case of Hodgkin's disease presenting as a fulminant hepatic failure is reported. A 34-year-old man presented with an acute onset of liver failure characterized by jaundice, ascites, encephalopathy and bleeding diathesis. Chemotherapy was initiated, resulting in a dramatic improvement not only in the patient's level of consciousness, but also in prothrombin time. Unfortunately, he succumbed shortly after to disseminated candidiasis. A post-mortem needle liver sample revealed massive hepatocellular necrosis, but no liver infiltration by the neoplastic disease. We conclude that in Hodgkin's disease, involvement of the liver can be manifested as a syndrome of paraneoplastic fulminant hepatic failure. In such cases, liver transplantation is an absolute contraindication but urgent chemotherapy under antifungal surveillance can be life saving.
Adult↗
Endothelial cell transplantation.
After more than 20 years of autologous endothelial cell transplantation, controversy is slowly giving way to consensus. The ongoing discussion regarding the optimal methods of creating an endothelial layer on synthetic vascular prostheses has been replaced by the realization that both in vitro endothelialization with cultured venous endothelial cells and mixed microvascular sodding result in equilibrated luminal tissue layers covered by a persistant endothelium. Clinical trials with almost 200 in vitro endothelialized prostheses are in their 10th year, and patency results are distinctly better than in nonendothelialized prostheses, particularly in below-the-knee grafts.
Blood Vessel Prosthesis↗
Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience.
BACKGROUND: Clinical in vitro endothelialization was assessed for its ability to improve the long-term patency of prosthetic femoropopliteal bypass grafts. METHODS: Between June 1989 and May 1998, 100 patients received 113 in vitro endothelialized expanded polytetrafluoroethylene grafts (ePTFE). Bilateral implantations were performed in 13 patients. In phase 1 of the study, 24 patients received 27 endothelialized grafts and 16 patients received 17 untreated grafts. In phase 2, endothelialization was offered to all patients who did not have a suitable saphenous vein available. Phase 2 began in June 1993 and included 76 patients who received 86 endotheliazed ePTFE grafts. In all, 100 patients had autologous endothelial cells harvested from 4- to 5-cm segments of a subcutaneous vein. In phase 1, the external jugular vein was used. In phase 2, the cephalic vein was used. These cells were grown to first-passage mass cultures and were lined confluently onto 6-mm ePTFE grafts, pre-coated with fibrin glue. Patency assessment for Kaplan-Meier survivorship analysis was determined by using duplex sonography and angiography. RESULTS: In phase 1, the Kaplan-Meier method revealed a primary 9-year patency rate for 65% for the endothelialized group, versus 16% for the control group (log-rank test, P = .002; Wilcoxon test, P = .003). In phase 2, the 5-year primary patency rate for all in vitro endothelialized infrainguinal reconstructions was 68% (66% for above-the-knee grafts and 76% for below-the-knee grafts). CONCLUSIONS: Nine years of clinical in vitro endothelialization provided strong evidence that autologous endothelial cell lining improves the patency of small-diameter vascular grafts and that a cell culture-dependent procedure can be used in a clinical routine.
Aged↗