3H-thymidine Uptake Research Articles

Modification of gamma radiation induced response of peritoneal macrophages and splenocytes by Hippophae rhamnoides (RH-3) in mice

Alcoholic extract of Hippophae rhamnoides, RH-3, reported to render >80% survival against lethal whole body Co-60-gamma irradiation (10 Gy) in mice, was investigated for its immunostimulatory effects. In comparison with un-irradiated control, whole body irradiation did not reduce peritoneal macrophage counts at 24 h post-irradiation. RH-3 treatment (30 mg kg(-1) body weight) alone or 30 min before whole-body irradiation enhanced viable counts of macrophages significantly (P< or =0.05) compared with both un-irradiated control and irradiated groups. Whole-body irradiation reduced the number of viable splenocytes significantly (P<0.05) compared with un-irradiated control at 24 h post-irradiation. RH-3 treatment alone or before whole-body irradiation appreciably countered radiation-induced decrease in splenocyte count. 3H-thymidine uptake method revealed that whole-body irradiation reduced splenocyte proliferation significantly (159 +/- 45 counts min(-1)/10(6) cells; P< or =0.05) in comparison with control (607 +/- 142 counts min(-1)) at 24 h after irradiation but RH3 treatment before irradiation reduced the steep decrease and maintained it as 444+/-153 counts min(-1). After whole-body irradiation, the ratio of spleen weight/mouse weight decreased to 1.5 +/- 04 compared with 2.9 +/- 0.32 in un-irradiated control at 24 h post-irradiation. Similarly, total protein content in splenocytes also decreased to 48 +/- 6 microg/10(6) cells in comparison with 368 +/- 16 microg/10(6) cells of un-irradiated control. RH-3 treatment before irradiation countered radiation-induced decrease in both spleen weight/mouse weight ratio (4.0 +/- 0.35) and total protein content (360 +/- 13 mug/10(6) splenocytes). In the supernatant of peritoneal macrophage cultures exposed to 2 Gy Co-60-gamma radiation ex-vivo, the total nitrite content was enhanced significantly (P<0.05) to 5.72 +/- 0.09 microM in comparison with un-irradiated control (1.64 +/- 0.09 microM). RH-3 treatment (30 microg mL(-1)) before irradiation reduced total nitrite significantly (0.93 +/- 0.3; P< or =0.05) in comparison with irradiated control group. At 24 h after whole body irradiation, the CD4+/CD8+ ratio reduced to 1.5 in comparison with un-irradiated control (1.9) but RH-3 treatment before irradiation restored the ratio to 2.1. These findings explicitly reveal the immunostimulatory activity of RH-3, which may play an important role in the manifestation of its radioprotective efficacy.

Autoantibodies and Cell‐mediated Autoimmunity to NMDA‐type GluRɛ2 in Patients with Rasmussen's Encephalitis and Chronic Progressive Epilepsia Partialis Continua

To evaluate antibody-mediated and cytotoxic T cell-mediated pathogenicity that has been implicated as the autoimmune pathophysiological mechanism in Rasmussen's encephalitis. We examined autoantibodies against the N-methyl-d-aspartate glutamate receptor (NMDA-type GluR) epsilon2 subunit and its epitopes in serum and CSF samples from 20 patients [five histologically proven (definitive) Rasmussen's encephalitis with epilepsia partialis continua (EPC), four definitive Rasmussen's encephalitis without EPC, and 11 clinical Rasmussen's encephalitis with EPC]. We examined 3H-thymidine uptake into lymphocytes after stimulation by GluRs. All nine definitive patients (five patients with EPC and four without EPC), and 10 of 11 clinical Rasmussen's encephalitis patients had the autoantibodies. In four patients, the autoantibodies were absent in early stage when epileptic seizures had already become frequent, and appeared subsequently. In two patients, the autoantibodies persisted in the serum after frontal lobe resection or functional hemispherectomy, although epileptic seizures were completely controlled. Autoantibodies to the C2 epitope predominated, while autoantibodies to the extracellular N epitope were rare. The mean 3H-thymidine uptake ratios (stimulation by GluRepsilon2-containing homogenates/stimulation by PHA) were significantly higher in definitive and clinical Rasmussen encephalitis patients than in controls. The mean 3H-thymidine uptake ratios (relative to PHA) were significantly higher for GluRepsilon2-containing homogenate than for control homogenate or GluRdelta2-containing homogenate. Autoantibodies against GluRepsilon2 may be one of the diagnostic markers for Rasmussen's encephalitis with and without EPC. Patients have activated T cells stimulated by GluRepsilon2 in peripheral blood circulation. We speculate that cellular autoimmunity and the subsequent humoral autoimmunity against GluRepsilon2 may contribute to the pathophysiological processes in Rasmussen's encephalitis.

A selective cyclic integrin antagonist blocks the integrin receptors αvβ3 and αvβ5and inhibits retinal pigment epithelium cell attachment, migration and invasion

BackgroundProliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors αvβ3 and αvβ5, was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins αvβ3 and αvβ5 on RPE cells was examined.MethodsThe effect of a cyclic integrin antagonist and a control peptide (0.01 μg/ml to 300 μg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H3-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors αvβ3 and αvβ5 was evaluated by flow cytometry.ResultsThe integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1–10 μg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3–10 μg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1–10 μg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3μg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors αvβ3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and αvβ5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold).ConclusionA selective inhibition of the integrin receptors αvβ3 and αvβ5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

Results of a placebo-controlled phase III trial of immunotherapy with APC8015 for patients with hormone refractory prostate cancer (HRPC)

4500 Background APC8015 is an immunotherapy cellular product consisting of autologous peripheral blood mononuclear cells enriched for a dendritic cell fraction pulsed with PA2024, a Prostatic Acid Phosphatase (PAP)-GM-CSF construct. We previously reported results including time to objective progression (TTP) and time to onset of disease related pain (TDRP) from a randomized trial in metastatic HRPC. Reported here are final overall survival (OS) data from this trial, as well as results of immunologic testing. Methods Patients (pts) with asymptomatic, metastatic HRPC were randomized (2:1) to receive APC8015 (n=82) or placebo (n=45) every 2 weeks x 3. Eligible pts had > 25% of cancer cells positive for PAP by central pathology review. The primary endpoint was TTP. TDRP was a secondary endpoint and pts were monitored for OS at 36 months. Monitoring was by weekly pain logs and serial radiologic imaging. Progression and pain were centrally reviewed and pts were followed per protocol for survival for 3 years following randomization. T-cell proliferation to PA2024 was evaluated by 3H-thymidine uptake. The T-Cell Stimulation Index (TCSI) was defined as: counts per minute (cpm) with antigen/cpm without antigen. The TCSI-ratio was defined as the median TCSI at 8 weeks/median TCSI at pre-treatment. Results 127 pts were randomized between 1/00 and 10/01. In an intent-to-treat (ITT) analysis, median OS was 25.9 months for pts on APC8015 compared with 22.0 months for those on placebo (p=0.020, log-rank, hazard ratio 1.625). At 36 months, 33% of APC8015 pts were alive, compared with 11% of placebo pts. The TCSI-ratio was 16.9 for APC8015 and 1.99 for placebo (p=.003, Wilcoxon Rank Sum). Treatment was generally well tolerated. Grade 1 and 2 fevers and rigors were the most common treatment-related AEs. Conclusions In an ITT analysis, treatment with APC8015 resulted in a statistically significant OS advantage of 3.9 months in pts with asymptomatic HRPC, representing the first survival advantage attributed to an immunotherapy product in prostate cancer. APC8015 pts also had an 8-fold increase in the TCSI-ratio. These data support this approach in the treatment of pts with metastatic HRPC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Dendreon Dendreon Dendreon Dendreon Dendreon Dendreon

Outer Membrane Vesicles From Porphyromonas gingivalis Affect the Growth and Function of Cultured Human Gingival Fibroblasts and Umbilical Vein Endothelial Cells

The purpose of this study was to examine the effects of outer membrane vesicles (OMV) obtained from Porphyromonas gingivalis (Pg) on the growth and function of human gingival fibroblasts (HGF) and human umbilical vein endothelial cells (HUVEC). OMV were obtained from a cell-free growth medium of Pg ATCC 33277 by 40% NH2SO4 precipitation and ultracentrifugation. Cell proliferation was measured by 3H-thymidine incorporation into growing HGF and HUVEC. Endothelial cell function was determined by their capacity to form a network of capillary tubes on an extracellular matrix (ECM). Proliferating HGF and HUVEC demonstrated a significant dose-dependent inhibition of 3H-thymidine uptake when cultured with 0 to 40 microg/ml of OMV protein. HGF and HUVEC showed an IC50 of growth of about 9.0 microg/ml and 4.5 microg/ml of OMV protein, respectively. Capillary tube formation by HUVEC cultured on an ECM was suppressed by 70% to 80% with 5 microg/ml OMV protein after 18 hours of incubation. The presence of proteolytic enzymes in the OMV did not contribute to capillary tube disruption, since blocking enzyme activity with specific inhibitors did not reduce the suppression of capillary tube formation. After heating at 90 degrees C for 5 minutes, OMV significantly lost their capacity to suppress capillary tube formation. OMV significantly inhibit the proliferation of cultured HGF and HUVEC in a dose-dependent manner. OMV suppressed the capillary tube formation by cultured HUVEC. The factor(s) appeared to be a protein and not endotoxin because its inhibitory activity was markedly reduced by heat inactivation. These studies suggest that OMV contribute to chronic periodontitis by suppressing cell proliferation and revascularization in periodontal tissues.

Anthrax tumor toxin (LeTx) selectively kills B-RAF mutant melanomas

7533 The efficacy of anthrax tumor toxin (LeTx) was tested on a panel of 18 human melanoma cell lines using a 3H-Leucine incorporation inhibition assay. 11/18 cell lines were sensitive to LeTx (IC50 < 350 pM, percent cell kill > 75%), while the remaining 7/18 were not sensitive to LeTx (IC50 > 750 pM, Percent cell kill < 60%). 10/11 sensitive melanoma cell lines carried the V599E BRAF. Only 1/7 resistant cell line was BRAF mutation positive (p < 0.0001). 6/7 resistant cell lines carried the Q61R or Q61K N-Ras mutation. Next, LeTx effects on 3H-Thymidine incorporation were measured. In 11/11 sensitive cell lines, the IC50 for 3H-thymidine incorporation was lower than that of 3H-Leucine incorporation indicating that the majority of surviving cells at maximal concentration were in cell cycle arrest. 4/7 cell lines that were not sensitive to LeTx by 3H-Leucine incorporation had low IC50 values in the 3H-Thymidine incorporation inhibition assay (IC50 < 275 pM) and a significantly lower percentage of 3H-thymidine uptake at maximum concentration suggesting significant tumor cell growth arrest rather than tumor cell death. Anthrax toxin receptor (ATR) levels, measured by saturation assay with 125I-PA, varied between 3562 and 32440 receptors/cell with resistant cell lines having the same levels of ATR expression as sensitive cell lines and no correlation of ATR receptor levels with IC50 for LeTx sensitive cell lines. Total and phosphorylated levels of MEK1/2 and ERK1/2 in the cell lines were quantified by western blots and densitometry. LeTx killing correlated with MEK1/2 activation levels. 9/11 sensitive melanoma cell lines and 2/7 resistant melanoma cell lines had a P-MEK1/2 to MEK1/2 ratio > 0.3 (p < 0.001). P-ERK1/2 to ERK1/2 levels did not correlate with melanoma cell line sensitivity to LeTx. The small molecular weight MEK1/2 inhibitor U0126 was tested for effects on the cell lines. 6/6 LeTx resistant cells were resistant to U0126; 5/9 LeTx sensitive cells were sensitive to U0126. In summary, melanoma cell line LeTx sensitivity correlated with B-Raf mutation, P-MEK1/2 levels, and U0126 sensitivity, but not ATR or P-ERK1/2. In future clinical trials of LeTx in metastatic melanoma, these results may provide molecular targets to identify responders. No significant financial relationships to disclose.

The detection of anti-erythropoietin antibodies in human serum and plasma: Part II. Validation of a semi-quantitative 3H-thymidine uptake assay for neutralizing antibodies

Neutralizing antibodies to erythropoietin (EPO) can cause a loss of response to recombinant human EPO (rHuEPO) and lead to rare cases of sudden, unexplained, severe anemia in chronic renal failure patients treated with rHuEPO. An assay for neutralizing anti-EPO antibodies has been validated that is based on the inhibition of proliferation of human UT-7/EPO cells, an immortalized cell line, by neutralizing antibodies in serum test samples using 3H-thymidine as a marker for proliferation. The dependence of the human cell line on EPO for growth and proliferation in a concentration-dependent manner enabled the validation of a rHuEPO standard curve for cell proliferation that can be used to determine the presence of neutralizing anti-EPO antibodies in serum samples. Proliferation of the cells increases with increasing concentrations of EPO, forming an S-shaped standard curve, which is fit with a 4-parameter logistic model, between 2.5 and 50 mU/mL rHuEPO, with a percent coefficient of variation (% CV) from 8.7% to 22.1% and a % accuracy of 103.5% to 109.5%. Anti-EPO antibodies and serum with anti-EPO antibodies neutralize UT-7/EPO proliferation by 10 mU/mL rHuEPO in a concentration- or dilution-dependent manner with < or = 25% CV. Percent neutralization is calculated by determining the amount of EPO recovered from the original 10 mU/mL added using the formula [((10-concentration recovered)/10)x100%]. Stem cell factor (SCF) stimulated cell proliferation, but not as effectively as rHuEPO. Antibodies to SCF were not able to inhibit the proliferative response induced by EPO and vice versa, confirming the specificity of the assay for antibodies to EPO. High EPO levels can impact both the radioimmunoprecipitation and neutralization assays to produce a false negative result. However, the impact can be mitigated by the large dilutions used in the neutralization assay.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive alpha-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating (3)H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating (3)H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

Coculture with dendritic cells promotes proliferation but not cytotoxic activity of γ/δ T cells

T cells bearing the γ9/δ2 T cell receptor (TCR) have recently raised interest as non-MHC restricted effector cells against multiple myeloma. They are described to be stimulated by phosphoantigens without the need of antigen presenting cells. However, in the past a positive effect of cells of the monocyte lineage on activation of γ/δ T cells has been shown. Monocyte derived dendritic cells (DC) are professional antigen presenting cells widely investigated as stimulators of α/β T cells. But only little is known about the interaction of γ/δ T cells and monocyte derived DC. Here, we investigated the effect of coculture of mature DC unpulsed or pulsed with ibandronate on the proliferation and cytotoxic activity of isolated γ/δ T cells. After coculturing monocyte derived DC with isolated γ/δ T cells, proliferation of γ/δ T cells was enhanced as determined by the 3H thymidine uptake assay. Also, IFN-γ secretion was increased after coculture with DC. As DC are well known to induce activation of α/β T cells we investigated whether the cytotoxic activity of γ/δ T cells could be increased by coculture with DC. We found no difference in cytotoxic activity of γ/δ T cells alone or cocultured with unpulsed or pulsed mature DC. Also, sensitizing of myeloma cells by addition of ibandronate could not increase lysis by γ/δ T cells. In conclusion, monocyte derived DC are capable of stimulating proliferation and secretion of IFN-γ of γ/δ T cells but do not exert an effect on cytotoxic activity of γ/δ T cells against myeloma cells.

Primary production and microbial activity in the euphotic zone of Lake Baikal (Southern Basin) during late winter

Three years of regular weekly/biweekly monitoring of seasonal changes in temperature, transparency, chlorophyll a (CHL) and bacteria [erythrosine-stained microscopic counts and cultivable colony forming units (CFUs)] at the vertical profile in the South basin of Lake Baikal (51°54′195″N, 105°04′235″E, depth 800 m) were evaluated. In more detail, the structure and function of phytoplankton and the microbial loop in the euphotic layer at the same site were investigated during the late-winter–early-spring period under the ice. The depth of euphotic zone (up to 1% of surface irradiation) was 35 to 40 m. Primary production was measured three times a week with the 14C method in 2, 10, 20, 30 and 40 m. Maximum production was found in 10 m, with lower values towards the surface (light inhibition) and towards the lower layers. The total production in cells larger than 1 μm in the column (0–40 m) was 204–240 mg C d −1 m −2, 30–40% of it being in cells 1–3 μm (mostly picocyanobacteria), which represented roughly 9% of the total chlorophyll a (estimated from pigment analyses). A major part of phytoplankton biomass was formed by diatoms ( Synedra acus Hust., Asterionella formosa Hass. and Stephanodiscus meyerii Genkal & Popovskaya). Total production (including extracellular, dissolved organic matter) was 235–387 mg C day −1 m −2, and the exudates were readily used by bacteria (particles 0.2–1 μm). This part amounted to 1–5% of cellular production in 2 to 20 m and 11–77% of cellular production in 20–40 m, i.e., in light-limited layers. From 0 to 30 m, chlorophyll a concentration was 0.8 to 1.3 μg l −1, wherefrom it decreased rapidly to 0.1 μg l −1 towards the depth of 40 m. Bacteria (DAPI-stained microscopic counts) reached 0.5–1.4×10 6 ml −1; their cell volumes measured via image analysis were small (average 0.05 μm −3), often not well countable when erythrosine stain was used. Bacterial biomasses were in the range of 6–21 μg C l −1. Numbers of colony forming units (CFUs) on nutrient fish-agar were c. 3–4 orders lower than DAPI counts. The amounts of heterotrophic protists were low, whereby flagellates reached 6 to 87 ml −1 and ciliates, 0.2–1.2 ml −1 (mostly Oligotrichida). Bacterial production was measured in the same depths as primary production using 3H-thymidine (Thy) and 14C-leucine (Leu) uptake. Consistently, bacterial abundances, biomasses, thymidine and leucine production were higher by 30–50% in layers 2, 10 and 20 m compared with that in the deeper 30 and 40 m, where cellular primary production was negligible. Leucine uptake in the deeper layers was even three times lower than in the upper ones. From the comparison of primary and bacterial production, bacteria roughly use 20–40% of primary production during 24 h in the layers 2 to 20 m.

Effects of Power Frequency Electromagnetic Fields on Growth of Germinating Vicia faba L., the Broad Bean

Experimental investigations were carried out to evaluate the effect of continuous and delayed exposure of power frequency electromagnetic fields at 5, 50 and 100 μT on germinating Vicia faba seedlings as a model system. These studies included physical parameters (length and girth of primary roots, number as well as length of lateral roots and imbibition), major biochemical constituents (total sugar, protein, and fat) and activities of important housekeeping enzymes (amylases, proteases, and lipase) at 2, 4, and 8 days of growth. Also, mitotic index and rate of DNA synthesis were studied at day 8 of growth. There was no significant change in physical parameters and major biochemical constituents between control and experimental groups. Also, the comparison between the control and experimental group of seeds showed that α-amylase activity significantly decreased at 5, 50 and 100 μT on day 2 and 4 of growth. β-amylase and protease (37○C & 50○C) showed a significant decrease in activity on day 2 and 4 of growth at 100 μT, whereas activity of lipase significantly decreased only on day 2 of growth at 100 μT. At day 8 of growth, all enzyme activities reverted back to the same as control. Also, there was a significant increase in mitotic index as well as 3H-thymidine uptake at 100 μT delayed exposure on day 8. The present study suggests that exposure to power frequency electromagnetic fields up to 100 μT on germinating seedlings does not cause any permanent damage since the initial alteration under the magnetic fields in some important housekeeping enzymes involved in the onset of seed germination were returened to control values on day 8 of growth. Also, the growth of the germinated seedlings was found to be enhanced by the application of power frequency magnetic fields (100 μT) as evidenced by mitotic index and 3H-thymidine uptake.

Pharmacokinetics and Pharmacodynamics of Low Dose Mycophenolate Mofetil in HIV-Infected Patients Treated with Abacavir, Efavirenz and Nelfinavir

The use of mycophenolate mofetil in combination with highly active antiretroviral therapy (HAART) has been proposed in order to inhibit HIV replication. Due to the low doses involved, pharmacokinetic-pharmacodynamic monitoring is recommended. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic monitoring of low doses of mycophenolate mofetil (0.25 g twice daily) in HIV-infected patients treated with HAART and after programmed discontinuation of HAART, in order to assess whether low doses of this immunosuppressive agent provide a biological effect. Mycophenolic acid (MPA) plasma levels (assessed by high-performance liquid chromatography) and the capacity of patients' sera to inhibit CEM cell line proliferation (assessed by (3)H-thymidine uptake) were measured post-dose at 0, 20, 40 minutes and 1, 2, 4, 6, 8, 10 and 12 hours in nine HIV-infected patients treated with a combination of abacavir, nelfinavir and efavirenz (HAART) and mycophenolate mofetil 0.25 g twice daily at days 7, 28, 120 and 150 (30 days without HAART) after the treatment initiation. A control group of eight patients was treated with HAART alone. In the 35 post-dose curves analysed, no differences were found in MPA levels between days 7, 28, 120 and 150: area under the plasma concentration-time curve - mean value 15.3 mg . h/L, range 10.4-24.4 mg . h/L; minimum plasma concentration - mean value 0.60 mg/L, range 0.20-4.67 mg/L; maximum plasma concentration mean value 2.60 mg/L, range 0.94-7.98 mg/L. Pretreatment patients' sera did not inhibit CEM proliferation. Post-treatment patients' sera inhibited CEM proliferation to <40% in 25 of 35 curves at 0 hours (six of nine patients), in 34 of 35 curves at 1 hour, in 32 of 35 curves at 2 hours, in 22 of 35 curves at 4 hours, and in 8 of 35 curves at 12 hours. The MPA level versus CEM proliferation inhibition had a concentration that produces 50% of the maximum drug effect (EC(50)) of 0.33 mg/L. Viral load at day 150 was >200 copies/mL in all control patients and in three of nine patients receiving mycophenolate mofetil. These three patients were the only ones repeatedly unable to inhibit pre-dose CEM proliferation to <40%. Mycophenolate mofetil pharmacokinetic profiles in HIV patients under HAART are not significantly different from those found in transplant patients. Sera from the majority of patients receiving low doses of mycophenolate mofetil inhibited lymphocyte proliferation during most of the inter-dose interval, despite low MPA plasma levels. For some patients, higher doses may be necessary: the capacity of sera to inhibit CEM proliferation may help to identify these patients.

Isolation and Immunomodulatory Effect of Flavonoids fromSyzygium samarangense

Sixteen flavonoids were isolated from the acetone extract of the leaves of Syzygium samarangense Merr. et Perry. The isolated flavonoids were evaluated for immunopharmacological activity. Human peripheral blood mononuclear cells (PBMC) were used as target cells, and cell proliferation was determined by 3H-thymidine uptake. Among them, (-)-strobopinin (2), myricetin 3-O-(2''-O-galloyl)-alpha-rhamnopyranoside (8), (-)-epigallocatechin 3- O-gallate (10) and myricetin 3-O-alpha-rhamnopyranoside (11) showed inhibitory potency on PBMC proliferation activated by phytohemagglutinin (PHA). The IC50 values of compounds 2, 8, 10, and 11 on activated PBMC proliferation were 36.3, 11.9, 28.9, and 75.6 microM, respectively. The inhibitory mechanisms may involve the blocking of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production, since compounds 2, 8, 10 and 11 reduced IL-2 and IFN-gamma production in PBMC in a dose-dependent manner.

Lack of effective T-lymphocyte response to the PAX3/FKHR translocation area in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) frequently contains the fusion transcription factor PAX3/FKHR. Therefore, clinical studies have been initiated to utilize the PAX3/FKHR translocation point area as a peptide vaccine against ARMS. Our study was directed at identifying antigenic T-lymphocyte epitopes at the PAX3/FKHR translocation point area. The peptide sequence surrounding the PAX3/FKHR translocation point was evaluated by MHC binding algorithms for potential T-lymphocyte antigenic epitopes (class I molecules HLA-A1, -A2 and -A3; class II molecules HLA-DR1, -DR4 and -DR7). Using in vitro techniques, dendritic cells loaded with PAX3/FKHR peptides were used to stimulate naive T-lymphocytes. T-lymphocyte activity was then evaluated by 51Cr release and 3H-thymidine uptake assays. Only one HLA-A3-restricted epitope was predicted by the algorithms. The peptide was prepared and tested for its ability to stimulate naive cytotoxic T-lymphocytes (CTLs). Unfortunately, the peptide was unsuccessful at stimulating naive CTL. However, induction of naive helper T-lymphocytes (HTL) to recognize and respond to the PAX3/FKHR translocation peptide was successful. Yet, this HTL peptide activity did not translate into recognition of PAX3/FKHR-containing ARMS tumor cells. It appears that the fusion area of PAX3/FKHR may not be a good source of antigenic anti-tumor peptide epitopes. These results raise serious concerns about the success and applicability of future peptide-based vaccine immunotherapy directed at the PAX3/FKHR translocation point.

Development of Cell Based Therapeutic Vaccines for the Treatment of Multiple Myeloma.

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We proposed that semi-autologous myeloma hybrid cells, formed by fusing malignant plasma cells with the human lymphoblastoid cell line, HMy2, would generate immortalised cell lines with enhanced immunogenicity for T cell responses, which could be used as potential therapeutic vaccines. The specific goals of the project were: to generate stable hybrid cell lines in vitro, by fusion of HMy2 with ex vivo tumour cells from patients with MM; to characterise the hybrid cells with respect to growth in culture, stability, and surface marker phenotype; and to investigate the ability of the hybrid cells to stimulate T cell immune responses in vitro as compared with parental tumour cells. The parent HMy2 cell line was fused with ex vivo myeloma plasma cells (purified from the marrow using CD138+ magnetic beads) from 5 patients, and chemically selected in HAT medium (to which HMy2 is sensitive), to form stable cell lines that grew continuously in culture. Efforts of growing myeloma cell lines from all of the patients were unsuccessful. The hybrid status of 4 of the 5 fused cell lines was confirmed by microsatellite testing using polymorphic loci on chromosomes X, 16q, 7q, 13q, 5q, 2p, 11p, 12p. Furthermore, the microsatellite analysis was checked after 3 months of continuous culture (approximately 40 passages), and all of the hybrids remained genetically stable over this period. Hybrid cells were analysed for expression of immunologically relevant accessory molecules using flow cytometry, in comparison with parent tumour cells and HMy2. The hybrids retained CD138 expression, although expression of CD56 was reduced in hybrids from two of three CD56+ myeloma cases. CD54 (ICAM-1) and CD50 (ICAM-3) were uniformly expressed in all hybrids. CD58 (LFA-3) was expressed more strongly in the hybrids than in original tumour cells, but CD11a (LFA-1) was weakly expressed or absent, in line with expression on HMy2 cells. All of the hybrids expressed CD40, CD80 (B7-1) and CD86 (B7-2), although CD80 was weakly or not expressed by myeloma plasma cells. Both MHC class I and II were expressed in all the hybrids, suggesting the potential for antigen presentation to both CD8+ and CD4+ T cells, in contrast to the majority of myeloma plasma cells, which failed to express MHC class II. The hybrid cells therefore expressed important markers to facilitate effective antitumour responses, which were absent on the parent tumour cells. The hybrid cells stimulated allogeneic T cell proliferative (3H thymidine uptake) responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, which failed to induce T cell proliferation above background levels. In addition, T cells from patients from whom hybrid cell lines had been derived responded to stimulation with both autologous and allogeneic hybrid cell lines, whilst failing to proliferate in response to autologous or allogeneic myeloma plasma cells. These data indicate that myeloma patients are immunocompetent, suggesting that active immunisation with semi-autologous hybrid cell lines represents a valid approach to immunotherapy in these patients. We have shown the generation of myeloma hybrid cell lines that grow continuously in tissue culture and have increased immunogenicity for T cell responses in vitro. This approach represents a novel strategy for the development of therapeutic vaccines for the treatment of multiple myeloma.

Generation of Human Regulatory T Cells Using NOD/SCID/γ CNULL Mice Model.

We have reported a NOD/SCID/γcnull (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment.Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3+ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4+ CD25+ cells have regulatory activity, CD4+ CD25− cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4+ CD25+ cells. The inhibition of proliferation by CD4+ CD25+ cells was analyzed by 3H-thymidine uptake. CD4+ CD25+ cells successfully suppressed the CD4+ CD25− T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4+ CD25+ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.

Ligand Activation of FLT3 Induces Cell Cycle Arrest in Acute Lymphoblastic Leukemia with 11q23 Translocation.

A fms-like kinase (FLT3) is widely known to be involved in proliferation of normal hematopoietic stem cells and precursors. Acute lymphoblastic leukemia (ALL) with 11q23 translocation, often found in infantile leukemia with very poor prognosis, is thought to be derived from a population of cells at the developmental stage very close to hematopoietic stem cells, and was recently shown that they express FLT3 at high levels compared with other types of leukemia. In the present study, we examined the effects of FLT3 ligand (FL) on leukemia cells with or without 11q23 translocation to evaluate a biological implication of the FLT3/FL interaction in ALL. Three of 8 leukemic cell lines without 11q23 translocation showed a proliferative response to FL in the 3H-thymidine uptake assays. However, five of 7 B-precursor leukemic cell lines with 11q23 translocation, unexpectedly, showed an inhibitory response (23-69% inhibition) to FL in a dose-dependent manner (1–20ng/ml), although the special cell line with D835 mutation in FLT3 (KOCL-33) was not affected by the addition of FL. This inhibitory effect was almost abrogated in the presence of a FLT-3 kinase inhibitor PKC412. Inhibition of 3H-thymidine uptakes were not due to induction of apoptosis but due to induction of the Go/G1 arrest. This cell cycle arrest was mediated, at least in part, by a marked up-regulation of p27 due to suppression of its degradation, and promoted resistance of cell lines to radiation-induced apoptosis. Of interest, the addition of FL induced a complete disappearance of constitutive phosphorylation of STAT5 but upregulated phosphorylation of MAPK and Akt. These results suggest that the FLT3/FL interaction in ALL with 11q23 translocation transmits the inhibitory signal specifically to the JAK/STAT pathway via the kinase activity of FLT3, in the process of which the JAK/STAT-specific inhibitory molecules such as SOCS-2 and CIS-1 may be implicated.

PKC412, a FLT3 Inhibitor, Effectively Induces Apoptosis of Leukemia Cells with 11q23 Translocation Particularly Having D835 Mutation.

About 30% cases of acute myeloid leukemia (AML) show FLT3 mutations such as internal tandem duplications within the juxtamembrane domain and point mutations within the kinase domain (D835 et al), which result in constitutive activation of FLT3 kinase activity. Although it was recently shown that acute lymphoblastic leukemia (ALL) with 11q23 translocation, often found in infantile leukemia with very poor prognosis, express FLT3 at high levels, few studies have been performed regarding the biological implication of FLT3 in leukemia with 11q23 translocation. We recently found that 2 of 6 cell lines with 11q23 translocation had D835 mutation in FLT3 (KOCL-33 with 11; 19 translocation and B-precursor phenotype, and KOCL-48 with 4; 11 translocation and monocytoid phenotype). In the present study, we evaluated the in vitro inhibitory effects of PKC412 (a kinase inhibitor of FLT3) using leukemic cell lines with or without D835 mutation. 3H-thymidine uptakes were inhibited by PKC412 at 50–200 nM very markedly in two 11q23-cell lines with D835 mutation (>90% inhibition), moderately (50–80% inhibition) in 11q23-cell lines without FLT3 mutation (n=5), but only modestly (<50% inhibition) in other B-precursor cell lines without FLT3 mutation (n=5). Two 11q23-cell lines with D835 mutation were effectively induced into apoptosis by PKC412, which was preceded by a prompt disappearance of constitutive phosphorylation of STAT5 before PKC412 treatment. The RNase protection assay revealed a decrease in mRNA of Bfl-1 which is known to be up-regulated by the NFkB activity, but showed no changes in mRNA of apoptosis-related molecules such as Bcl-2, Bcl-xl, Bad, Fas, FasL, and TRAIL. These results suggest that PKC412 is a potentially useful agent for the treatment of leukemia with 11q23 translocation, particularly having FLT3 mutation.

Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.

A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.