Radioactive Chromium Research Articles

Investigation of NK cell function against two target hematological cell line using radioactive chromium assay

NK (Natural killer) cells are a special population of peripheral blood lymphocytes that kill virus-infected cells as well as tumor cells. For testing NK cell function, the classic gold standard assay has been used for a long time, determining the activity from target tumor cells using radioactive chromium in cell cultures for 4h. In this study two hematological cell lines K562 and MDS where used and target and results showed different sensitivity to killing by NK cells separated from healthy volunteers. Results have been shown that MDS release significantly more radioactive chromium indicating higher degree of necrosis during cell culture. In addition, K562 cell line is better target for NK killing in all different E:T ratio in comparison to MDS cell line previously described. Based on this, it is suggested that K562 cells be continues used in the future as better target for investigation NK killing.

Limiting Immunogenicity of Diabetic Retinopathy Patient-Derived Induced Pluripotent Stem Cells by Knocking out Human Leukocyte Antigen and Overexpressing Cluster of Differentiation 47

Introduction: Diabetic retinopathy (DR), the most common complication of diabetes, is characterized by vision loss due to vascular endothelial cell damage of the retina. Notably, induced pluripotent stem cell (iPSC) therapy has shown promise in the regeneration of the retina after damage. A limitation of using these cells includes the risk of immune-rejection. The knockout of human leukocyte antigen (HLA) proteins prevents a host immune response to non-native cells; however, HLA depletion introduces natural killer (NK) cell-mediated responses. Overexpression of cluster of differentiation 47 (CD47) inhibits the activity of NK cells. This project aims to create a universal DR patient-derived iPSC platform whose immunogenicity is limited through genetic alterations. Methods: iPSCs will be reprogrammed from DR patient-derived fibroblasts and CRISPR-Cas9 will be used to knock out HLA and overexpress CD47. Alterations will be validated through Sanger sequencing, Western Blot and Immunofluorescence (IF) analysis. DR patient-derived iPSCs will be differentiated into endothelial cells to mimic the DR afflicted endothelial cells. These differentiated cells will then be co-cultured with NK cells and a cytotoxicity assay will be performed. Specifically, a chromium-release assay will be used by loading radioactive chromium into the genetically modified and unmodified endothelial cells and the chromium released by dying cells was monitored. Results: Compared to the unmodified cells, we expect less NK-mediated cell death for the genetically altered endothelial cells. Discussion: Effectively limiting the immunogenicity of the donor-derived iPSCs can establish a universal platform for future studies in DR therapy. Conclusion: The resulting donor-derived iPSCs can be used to test drug therapies for DR or new methods to repair blood vessel damage, among a multitude of new research.

Cell-Mediated Cytotoxicity Assays

Cell-mediated cytotoxicity measurements can be divided in two methods, depending on whether radioactive material is used or not. Natural Killer cell-mediated cytotoxicity is routinely measured with a short-term assay by labeling the target cells with radioactive chromium (51Cr). The advantages of this method are (1) highly sensitivity, (2) easy execution, (3) low spontaneous release, and (4) nontoxic markers. The disadvantages include short half-life of the label and handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/enzymatic assays are highly sensitive and easy to use but unfortunately works only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of the dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false positive results leading to a decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.

Abstract B57: NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC)

Abstract Introduction: Natural Killer (NK) Cell - and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally been assessed by the release of radioactive Chromium from target cells following lysis. This assay is laborious, and requires substantial quantities of patient blood to detect minor changes in cell lysis due to the inherent background noise (spontaneous release). We have developed an assay that can visualize individual target cells to detect cytolytic activity without involving radioactivity, via high-throughput imaging in microtiter well format. We also developed a miniaturized version of this assay using Terasaki plates to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor. Methods: The assay we developed, Target cell Visualization Assay (TVA™) images individual fluorescence-labeled target cells. Labelled K562 tumor cells were used as targets, and peripheral blood mononuclear cells (PBMC) as effector cells. In an assay setup similar to Chromium Release Assays (CRA), constant number of labelled target cells was incubated with serially diluted effector cells. Four hours later, the cells were transferred to flat bottom microtiter plates and the number of viable tumor cells was quantitated using a plate reader; CTL ImmunoSpot® Analyzer, S6ULT-00-9000. For the miniaturized version, one tenth of effector and target cells were plated in a Terasaki plate format and imaged directly in the assay wells. Results: Only viable target cells retain the fluorescent dye; it is lost upon cell death. When effector and target cells are mixed in various ratios, the % of target cell lysis is inversely proportional to the number of effector cells in the well. The TVA™ and CRA in a 96-well format provided equivalent results. However, the TVA™ is a non-radioactive system and can be performed with much less labor. The TVA™ assay exhibited high intermediate precision as well as inter-assay repeatability. The assay results obtained in Terasaki plates, miniaturizing the assay tenfold, paralleled those of the 96-well format. Conclusion: The miniaturized TVA™ uses only 100,000 effector cells for eight serial dilutions, for Effector:Target ratios starting from 100:1. We have demonstrated the feasibility of detecting and assessing NK function in a non-radioactive, high-throughput capable system using a fraction of the blood/effector cells as is required by traditional CRA. This is of particular value when access to PBMC is limited, such as pediatric, geriatric and immune deficient populations. The assay readout and analysis is automated by the CTL ImmunoSpot® software providing audit trails for each test condition. Citation Format: Srividya Sundararaman, Kinga Karacsony, Diana Roen, Jaya Ghosh, Paul V. Lehmann. NK cell-mediated cytotoxicity detection can be miniaturized by direct imaging in assay wells using a Terasaki plate format using only 100,000 effector cells (PBMC). [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B57.

Measuring Cytotoxicity by Bioluminescence Imaging Outperforms the Standard Chromium-51 Release Assay

The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

Modeling red blood cell survival data

Anaemia of chronic kidney disease (CKD) is a common complication in patients with renal impairment, especially in end-stage renal failure. As well as erythropoietin deficiency, decreased red blood cell survival is a contributing factor. However, it remains unclear which mechanism underlies the altered survival of red blood cells (RBCs). In this work a previously developed statistical model for RBC survival was applied to clinical data obtained from 14 patients with CKD undergoing hemodialysis as well as 14 healthy controls using radioactive chromium (⁵¹Cr) as random labelling method. A classical two-stage approach and a full population analysis were applied to estimate the key parameters controlling random destruction and senescence in the model. Estimating random destruction was preferred over estimating an accelerated senescence in both approaches and both groups as it provided the better fit to the data. Due to significant nonspecific random loss of the label from the cells that cannot be quantified directly only a relative RBC survival can be obtained from data using ⁵¹Cr as labelling method. Nevertheless, RBC survival was found to be significantly reduced in CKD patients compared to the controls with a relative reduction of 20-30% depending on the analysis method used.

Evaluation of red blood cell labelling methods based on a statistical model for red blood cell survival

The aim of this work is to compare different labelling methods that are commonly used to estimate the lifespan of red blood cells (RBCs), e.g. in anaemia of renal failure, where the effect of treatment with erythropoietin depends on the lifespan of RBCs. A previously developed model for the survival time of RBCs that accounts for plausible physiological processes of RBC destruction was used to simulate ideal random and cohort labelling methods for RBCs, as well as the flaws associated with these methods (e.g. reuse of label and loss of the label from the surviving RBCs). Random labelling with radioactive chromium and cohort labelling using heavy nitrogen were considered. Blood sampling times were determined for RBC survival studies using both labelling methods by applying the theory of optimal design. It was assessed whether the underlying parameter values of the model are estimable from these studies, and the precision of the parameter estimates were calculated. In theory, parameter estimation would be possible for both types of ideal labelling methods without flaws. However, flaws associated with random labelling are significant and not all parameters controlling RBC survival in the model can be estimated with good precision. In contrast, cohort labelling shows good precision in the parameter estimates even in the presence of reuse and prolonged incorporation of the label. A model based analysis of RBC survival studies is recommended in future to account for limitations in methodology as well as likely causes of RBC destruction.

Red Blood Cell Survival in Long-term Dialysis Patients

Shortening of red blood cell (RBC) survival contributes to the anemia of chronic kidney disease. The toxic uremic environment accounts for the decreased RBC life span. The contribution of mechanical damage caused by hemodialysis to the shortened life span is unclear. Reductions up to 70% in RBC survival have been reported in uremic patients. To date, no accurate well-controlled RBC survival data exist in dialysis patients treated using different dialysis modalities and receiving erythropoiesis-stimulating agent (ESA) therapy. The aim of this study was to determine RBC survival in hemodialysis (HD) and peritoneal dialysis (PD) patients compared with healthy persons. Observational study. 14 HD patients and 5 PD patients were recruited from the dialysis unit. Healthy volunteers (n = 14) age- and sex-matched to HD participants were included. All dialysis patients received either ESA therapy or regular iron supplementation. Dialysis patients versus age- and sex-matched healthy controls. RBC survival. RBC survival was determined using radioactive chromium labeling. More than 85% of dialysis patients were anemic (hemoglobin, 12.0 ± 1.1 g/dL); hemoglobin concentrations were not significantly different between HD and PD patients. Median RBC survival was significantly decreased by 20% in HD patients compared with healthy controls: 58.1 (25th-75th percentile, 54.6-71.2) versus 72.9 (25th-75th percentile, 63.4-87.8) days (P = 0.02). No difference was shown between the PD and HD groups: 55.3 (25th-75th percentile, 49.0-60.2) versus 58.1 (25th-75th percentile, 54.6-71.2) days (P = 0.2). Label loss from RBCs associated with the chromium 51 labeling technique needs to be accounted for in the interpretation of RBC survival data. Despite current ESA therapy, decreased RBC survival contributes to chronic kidney disease-related anemia, although the reduction is less than previously reported. There does not appear to be net mechanical damage associated with HD therapy resulting in decreased RBC life span.

Antipropulsive effects of central and peripheral morphine in the rat gastrointestinal tract.

The antipropulsive effects of centrally or peripherally administered morphine have been examined at three levels of the rat gastrointestinal tract. Adult male rats were anaesthetized with pentobarbitone (50 mg kg-1 i.p.) and were implanted with an intraluminal catheter in either the proximal duodenum or mid-jejunum. Other animals were also implanted with a cannula in the right lateral cerebral ventricle. Gastric emptying and transit were determined selectively by measuring the progression of a radioactive chromium (CR-51) solution, given intragastrically for tests of gastric emptying or instilled into the proximal or distal intestinal catheter for determination of intestinal transit, 30 min after administration of morphine or saline given either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.). Morphine given either s.c. (5 mg kg-1) or i.c.v. (30 micrograms, total dose) significantly inhibited gastric emptying and transit through bulk portions of the small intestine indicating that by either route it inhibits propulsion at all three levels of the gastrointestinal tract.

Blood volume and red cell mass in children with moderate and severe malaria measured by chromium‐53 dilution and gas chromatography/mass spectrometric analysis

Understanding blood volume changes in children with malaria is important for managing fluid status. Traditionally, blood/red cell volume measurements have used radioactive chromium isotopes. We applied an alternative approach, using non-radioactive chromium-53 labelling and mass spectrometry to investigate red cell volume (RCV) in Gabonese children with malaria. Nineteen children with malaria participated (10 severe, 9 moderately severe; ages 15 months to 7 years). Blood labelled with (53)Cr-chromate ex vivo was re-injected, then sampled 30 min later. Pre- and post-injection (53)Cr content were measured by gas chromatography/electron ionisation mass spectrometry of the chromium-trifluoroacetylacetone (TFA) chelate, calibrated against (50)Cr standards. Blood and red cell volumes were calculated from isotopic dilution in 15 of 19 children (in four, insufficient signal mitigated analysis). In this small pilot study, there were no significant differences between moderate and severe cases. Including all subjects, the mean RCV was reduced compared with predicted values (184 vs. 269 mL; p = 0.016) but blood volume, 71 +/- 33 mL/kg (normalised for weight), was close to predicted, approximately 77 mL/kg, commensurate with reduced haematocrit. Blood lactate concentration correlated negatively with RCV/weight (r = -0.56, p = 0.028), consistent with anaemia. In one case, sequential samples over 42 days gave an estimated rate of (53)Cr disappearance of 1.4%/day (equivalent half-life: 70 days). (53)Cr-labelling of red cells may be used to estimate blood and red cell volumes and can be used as an investigative tool in situations such as childhood diseases and resource-constrained settings. Although the red cell mass is depleted in malaria, the blood volume appears relatively well preserved.

Faecal blood loss with aspirin, nonsteroidal anti-inflammatory drugs and cyclo-oxygenase-2 selective inhibitors: systematic review of randomized trials using autologous chromium-labelled erythrocytes

IntroductionFaecal blood loss has been measured using autologous erythrocytes labelled with radioactive chromium for several decades, using generally similar methods. We conducted a systematic review of studies employing this technology to determine the degree of blood loss associated with use of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase-2 selective inhibitors (coxibs).MethodsA systematic search of PubMed and the Cochrane Library (to December 2006) was conducted to identify randomized trials in which treatment with aspirin, NSAIDs, or coxibs was continued for at least 7 days, and with at least 7 days of washout for crossover trials. Rates of faecal blood loss associated with these agents were determined in the randomized trials identified. Comparators were placebo, active, or no treatment. Outcomes of interest were mean daily faecal blood loss, and the number or proportion of individuals recording faecal blood above 5 ml/day and above 10 ml/day.ResultsForty-five reports of 47 trials were included, including 1,162 individuals, mostly healthy volunteers and predominantly young men. Only 136 patients (as opposed to healthy volunteers; 12%) were included, and these were mostly older people with an arthritic condition. Most NSAIDs and low-dose (325 mg) aspirin resulted in a small average increase in faecal blood loss of 1 to 2 ml/day from about 0.5 ml/day at baseline. Aspirin at full anti-inflammatory doses resulted in much higher average levels of blood loss of about 5 ml/day. Some individuals lost much more blood than average, at least for some of the time, with 5% of those taking NSAIDs having daily blood loss of 5 ml or more and 1% having daily blood loss of 10 ml or more; rates of daily blood loss of 5 ml/day or 10 ml/day were 31% and 10%, respectively, for aspirin at daily doses of 1,800 mg or greater.ConclusionAt baseline, or with placebo, faecal blood loss is measured at 1 ml/day or below. With low-dose aspirin and some NSAIDs, average values may be two to four times this, and anti-inflammatory doses of aspirin result in much higher average losses. A small proportion of individuals respond to aspirin or NSAIDs with much higher faecal blood loss of above 5 ml/day or 10 ml/day. There are significant limitations regarding the quality and validity of reporting of these studies, such as limited size and inclusion of inappropriate participants. The potential for blood loss and consequent anaemia requires more study.

Immune Responses to Guided Imagery During Breast Cancer Treatment

The use of relaxation and guided imagery to reduce stress and improve immune function has great potential benefits for patients with breast cancer. This pilot study used a pretest-posttest experimental design with 28 breast cancer patients, aged 25 to 75 years, with the diagnosis of stage 0, 1, or 2 breast cancer. The experimental group received a relaxation and guided imagery intervention and the control group received standard care. The effects of the intervention on immune function were measured by natural killer (NK) cell cytotoxicity and IL-2-activated NK cell activity prior to surgery and 4 weeks postsurgery. NK cell activity was measured using a 15-hr incubation chromium release assay. Cytotoxicity of NK cells was measured against chromium-labeled K-562 target cells. IL-2 was used to enhance reactivity of NK cells against tumor cells. After incubation for 15 hr, cytotoxicity was measured through the release of radioactive chromium. Significant differences between groups were found at 4 weeks postsurgery. T-tests showed increased NK cell cytotoxicity for the intervention group at 100:1, 50:1, and 25:1 effector cell: target cell ratios (E:T) (p < .01 to p < .05) and increased activation for IL-2 at 100:1, 50:1, 25:1, and 12.5:1 (E:T) (p < .01 to p < .05) for the intervention group as compared to the control group. These findings suggest that a relaxation intervention such as guided imagery could have an effect on NK cell cytotoxicity and NK cell cytotoxicity after activation with IL-2 in patients undergoing surgery for breast cancer.

Inclusion of an S-nitrosylating agent in the insufflating gas does not alter gastric activity in rats following pneumoperitoneum

We have previously shown that mixing the S-nitrosylating agent ethyl nitrite with carbon dioxide can attenuate pneumoperitoneum-induced decreases in splanchnic blood flow, but it was unclear if this agent would alter gastric function. This question was answered using rats by assessing gastric emptying and gastrointestinal transit times following gavage with radioactive chromium. There were five experimental groups: absolute control, anesthesia control, and carbon dioxide alone or with 100 or 300 parts per million ethyl nitrite. The period of insufflation was 1 h, and all animals were euthanized 6.5 h after chromium administration. The mean amount of radioactivity remaining in the stomach ranged between 16% and 27% of the total administered; these differences were not statistically significant (p > 0.05). Modest differences in chromium distribution were identified in the gastrointestinal tract, but for all treatments, the peak amount of radioactivity was located in the distal portion. Location of the peak, expressed as a percentage of total tract length, varied between 70% and 85% (p = 0.366). This study found no adverse effect of ethyl nitrite on postoperative gastric emptying or gastrointestinal transit time following pneumoperitoneum. The findings support continued assessment of the clinical utility of ethyl nitrite in the setting of laparoscopic surgery.

The effect of vagotomy and drainage operations on the rate of gastric emptying in duodenal ulcer patients

Abstract The rate of gastric emptying was estimated in 29 patients with duodenal ulcers, before and after vagotomy and drainage operations, by measuring the disappearance from the stomach of a standard meal containing radioactive chromium. When Heineke-Mikulicz pyloroplasty accompanied vagotomy gastric emptying was observed to be slowed, whereas after vagotomy and Fitmey pyloroplasty the rate of gastric emptying was more rapid when compared with the preoperative rate. Gastric emptying after vagotomy and gastrojejunostomy showed no consistent alteration in rate. Temporary gastric stasis occurred in the early postoperative period after vagotomy and drainage procedures.

Quantifying the Stress Induced by Distress in Patients With Lumbar Disc Herniation in Terms of Natural Killer Cell Activity Measurements

A comparative study was conducted. To attempt quantification of the stress in patients with lumbar disc herniation by measuring natural killer cell activity, and to evaluate the assay methodology. Stressful events may have adverse effects on health. Distress in the patient with lumbar disc herniation also may be a stressful event. Previous findings have shown the activity of natural killer cells to be impaired in medical student volunteers and after bereavement or other stressful life events. However, the stress in patients with spinal disorders has not been evaluated. The standard assay for natural killer cell activity has been the chromium release assay. However, this assay requires the use of radioactive chromium. Flow cytometric assay is a nonradioactive method that analyzes the activated natural killer lymphocyte subsets: CD45+, CD56+, CD69+. Peripheral blood lymphocytes were obtained from 20 patients with lumbar disc herniation (mean age, 30.8 years) and 20 healthy individuals (mean age, 38.9 years). Both chromium release assay and flow cytometric assay were performed for each patient. The mean natural killer cell activity in the patients (8.0% +/- 4.3%) was significantly lower than in the 20 healthy adult volunteers (12.6% +/- 4.5%). The mean natural killer cell activity in the healthy volunteers was 12.6% +/- 4.5% for the chromium release assay and 11.0% +/- 4.1% for the flow cytometric assay. The correlation (r = 0.77) of natural killer cell activity between the two methods was high. Natural killer cell activity in the patients with lumbar disc herniation was significantly lower than in healthy individuals, and the stress induced by pain and other distress may be present in patients with lumbar disc herniation. For measuring natural killer cell activity, flow cytometric assay is a reproducible assay, and it may be a viable alternative to the standard chromium release assay.

Interleukins 2 and 12 Produce Recovery of Cytotoxic Function in Tributyltin-Exposed Human Natural Killer Cells

Cytotoxic function of human natural killer (NK) cells is modulated by a variety of cytokines. Interleukins (IL) 2 and 12 are both potent stimulators of NK cell cytotoxic function. Tributyltin (TBT) is used in a variety of consumer products and industrial applications. TBT is found in dairy products, meat, and fish. We and others have shown that there are measurable levels of TBT in human blood. Butyltins appear to increase the risk of cancer and viral infections in exposed individuals. We have demonstrated that the ability of NK cells to kill tumor cells is greatly diminished after a l-h exposure to TBT and that this inhibition persists even after removal of the compound. In the current study we examine the effects of the NK-stimulatory ILs, IL2 and IL12, on the ability of NK cells to recover from the persistent inhibitory effects of a 1-h TBT treatment. Highly purified NK cells (>95% CD16+) or a lymphocyte preparation containing both T lymphocytes and NK cells were treated with 300 nM TBT and then allowed to recover for 24 h, 48 h, 4 days, and 6 days in TBT-free media containing no interleukin, 1000 U/mL IL2, 20 ng/mL IL l2, or a combination of IL2 plus IL12. Tumor killing function was then tested using a radioactive chromium release assay. As seen in our previous studies there is no recovery of NK cell cytotoxic function even after a 6-day recovery period when no interleukin is present in the medium. However, there is significant recovery of NK cytotoxic function when IL2, IL12, or the combination of IL2 plus IL12 is present in the medium during the recovery period.

Interleukins 2 and 12 Produce Significant Recovery of Cytotoxic Function in Dibutyltin-Exposed Human Natural Killer Cells

Cytotoxic function of human natural killer (NK) cells is modulated by a variety of cytokines. Interleukins (IL) 2, 12, 15, and 18 and Interferon γ (IFNγ) are potent stimulators of NK cell cytotoxicity. Butyltins (BTs) are used in a variety of consumer products and industrial applications. Dibutyltin (DBT) is found in plastic products, beverages stored in PVC pipes during manufacturing, and poultry products. BTs appear to increase the risk of cancer and viral infections in exposed individuals. Recently, we have demonstrated that the ability of NK cells to kill tumor cells is greatly diminished after a 1-h exposure to dibutyltin. This inhibition of tumor killing function continues even after removal of the compound. There is no significant recovery of NK cytotoxic function even when the cells are allowed to recover for 6 days. In the current study we examine the effects of NK-stimulatory cytokines on the ability of NK cells to recover from the inhibitory effects of a 1-h DBT treatment. Highly purified NK cells (>95% CD16+) or a lymphocyte preparation containing both T lymphocytes and NK cells were treated with 5 μM DBT and then allowed to recover for 24 h, 48 h, 4 days, and 6 days in DBT-free medium containing either no cytokine or a maximally stimulatory dose of several NK-stimulatory cytokines. Tumor killing function was tested using a radioactive chromium release assay. As seen in our previous studies there is no recovery of NK cell cytotoxic function even after a 6-day recovery period when no cytokine is present in the medium. However, there is significant recovery of NK cytotoxic function when IL2, IL12, or the combination of IL2 plus IL12 is present in the medium during the recovery period. The other cytokines tested (IL15, IL18, and IFNγ) were unable to increase the cytotoxicity of DBT-exposed NK cells.

The immune haemolytic anaemias: a century of exciting progress in understanding.

The immune haemolytic anaemias: a century of exciting progress in understanding.

Vascular Endothelial Cells Generate Peroxynitrite in Response to Carbon Monoxide Exposure

Carbon monoxide causes a perivascular oxidative injury in animals, and we tested the hypothesis that endothelial cells could be a source of the injurious oxidants. Studies were undertaken to assess whether exposure to carbon monoxide would cause cultured bovine pulmonary artery endothelial cells to liberate reactive species. Concentrations of carbon monoxide between 11 and 110 nM caused progressively higher concentrations of nitric oxide to be released by endothelial cells based on measurements of nitrite and nitrate. Intracellular production of peroxynitrite was indicated by elevated concentrations of nitrotyrosine, and extracellular liberation of peroxynitrite was indicated by oxidation of p-hydroxyphenylacetic acid and dihydrorhodamine-123. Carbon monoxide did not disturb mitochondrial function based on the rate of oxygen consumption, intracellular production of hydrogen peroxide, and the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Carbon monoxide also did not alter arginine transport by cells or nitric oxide synthase activity, but it was found to increase steady state levels of nitric oxide by competing for intracellular binding sites. Acute cytotoxicity from carbon monoxide, assessed as radioactive chromium leakage, was due to nitric oxide-derived oxidants. A delayed cell death, whose mechanism is not entirely clear, was also demonstrated by chromium leakage and uptake of vital stain. These findings offer a possible mechanism for adverse health effects caused by carbon monoxide at concentrations ranging from the relatively low levels in polluted environments to levels typically encountered with life-threatening poisoning. Carbon monoxide causes oxidative stress by a novel mechanism involving a competition for intracellular binding sites which increases steady state levels of nitric oxide and allows for generation of peroxynitrite by endothelium.

Generation of therapeutic T cells from draining lymph nodes in a murine model of head and neck squamous cell carcinoma.

To study immunotherapy for advanced head and neck squamous cell carcinoma using AT-84, a spontaneously arising murine tumor. We examined the draining lymph nodes (DLNs) for generation of potential therapeutic lymphocytes in head and neck squamous cell carcinoma. Experimental randomized control trial. Therapeutic T cells from DLNs were generated by the sequential activation of the in vivo-primed DLN cells with 2C11, and anti-CD3 antibody, and interleukin-2 (IL-2). Immunotherapy of mice bearing lung metastases was carried out in various experiments with low-dose systemic IL-2 and activated DLN cells. Using a 4-hour radioactive chromium Cr 51 release assay, in vitro cytotoxicity of these cells also was examined. Mice immunized with this tumor failed to reject the growth of a subsequent challenge with the tumor. Immunotherapy with low-dose systemic IL-2 resulted in a mean reduction of 79% in the number of lung metastases. Adoptive immunotherapy with activated DLN cells was effective in all experiments, with a mean reduction of 59% in the number of metastases in immunodeficient mice. However, DLN cells were not directly cytotoxic to the tumor cells in in vitro assays, unlike control lymphokine-activated killer cells. AT-84 is a nonimmunogenic tumor similar to many human malignant neoplasms, making this a suitable model for immunotherapy. Low-dose systemic IL-2 was effective in reduction of established metastasis in this model. Activated DLN cells show reproducible in vivo therapeutic efficacy despite lack of in vitro cytotoxicity. Use of DLN cells as sources of therapeutic T cells in patients with head and neck squamous cell carcinoma deserves exploration because they are readily obtainable and because conventional treatment is of limited benefit.