2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

18FDG, [18F]FLT, [18F]FAZA, and 11C-methionine are suitable tracers for the diagnosis and in vivo follow-up of the efficacy of chemotherapy by miniPET in both multidrug resistant and sensitive human gynecologic tumor xenografts.

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG), (11)C-methionine, 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT), and [(18)F]fluoroazomycin-arabinofuranoside ((18)FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive (Pgp(+)) and negative (Pgp(-)) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp(+) and Pgp(-) A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp(+) tumors. Our results demonstrate that (18)FDG, (18)F-FLT, (18)FAZA, and (11)C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacy of tumor chemotherapy in both Pgp(+) and Pgp(-) human tumor xenografts by miniPET.

The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

¹8FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein.

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

Metastatic hepatocarcinoma he/de tumor model in rat.

UNLABELLED: The aim of this study is to select among potential tumor models that could be suitable to follow the metastatic spead of tumor cells. (18)FDG-PET tumor diagnostic test has been adapted to investigate tumor growth in vivo in local and metastatic rat models. Materials and Methods. The expression of glucose transporters was traced by immunohistological analysis, followed by the uptake of (18)FDG and visualized by MiniPET scanner. After s.c. administration of hepatocarcinoma (He/De) cells intensive local tumor growth and (18)FDG uptake were measured. RESULTS: Whole body (18)FDG-PET imaging supported by histological analysis have shown that subcutaneously growing tumors did not project metastases to other sites from the injected area. To avoid local tumor formation i.v. injection was chosen, but did not improve the safety of tumor cell administration. Tumor formation after i.v. injection took a longer time than after s.c. administration. Tumors upon i.v. generation were smaller and detectable in liver and lung, but not in other organs or tissues. iii) Subrenally implanted He/De cells spread from the retroperitoneal primary tumor of the kidney to thoracal paratymic lymph nodes (PTNs). The spread from primary site to metastatic tumors in PTNs was confirmed by post mortem surgery and histological examinations. CONCLUSION: Among the three methods applied: a) Local s.c. administration of tumor cells generated local tumors unsuitable to study metastasis. b) Intravenous administration causing unpredicatable location of tumor formation is not regarded a reliable metastatic tumor model. c) Subreanal implantation model proved to be a suitable model to follow the metastatic process in rats.

Tumor-specific localization of self-assembled nanoparticle PET/MR modalities.

AIM: The aim of this work was to synthesize and study in vitro and in vivo nanocarriers used as magnetic resonance imaging (MRI) contrast agents that accumulate in tumor cells specifically overexpressing folate receptors. MATERIALS AND METHODS: Nanoparticles were prepared by self-assembly of poly-γ-glutamic acid and chitosan biopolymers and were complexed with gadolinium ions. Folic acid served as a targeting molecule. Rat hepatocellular carcinoma (HeDe) cells overexpressing folate receptors were used as a model system. For in vivo experiments, HeDe cells were transplanted under the renal capsule of F344 rats. RESULTS: In vitro results showed the significant internalization of nanoparticles into HeDe cells. MRI measurements revealed that targeting nanocarriers accumulated in tumors. The MRI/PET fusion images resulted in the exact localization of tumors. CONCLUSION: The nanocarrier provides a suitable means for the early diagnosis of tumors based on their overexpression of folate receptors.

Body fat distribution and metabolic consequences - Examination opportunities in dogs.

The relationship between metabolic disorders and the distribution of fat in different body regions is not clearly understood in humans. The aim of this study was to develop a suitable method for assessing the regional distribution of fat deposits and their metabolic effects in dogs. Twenty-five dogs were subjected to computed tomographic (CT) imaging and blood sampling in order to characterise their metabolic status. The different fat areas were measured on a cross-sectional scan, and the animals' metabolic status was evaluated by measuring fasting glucose, insulin and leptin levels. The volume of visceral adipose tissue is the main determinant of leptin levels. The correlation of visceral fat volume and leptin concentration was found to be independent of insulin levels or the degree of insulin resistance. There was a positive correlation between the visceral to subcutaneous fat volume ratio and serum insulin concentration, and a similar trend was observed in the relationship of fat ratio and insulin resistance. The distribution of body fat essentially influences the metabolic parameters in dogs, but the effects of adiposity differ between humans and dogs. The findings can facilitate a possible extrapolation of results from animal studies to humans with regard to the metabolic consequences of different obesity types.

In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF).

The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 µg·kg⁻¹ daily, for 2 weeks. Even at the highest concentration--a concentration highly toxic in vitro for all affected molds used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy.

Cancer cell targeting and imaging with biopolymer-based nanodevices.

We report here the synthesis, in vitro and in vivo investigation of magnetic resonance imaging (MRI) active nanoparticles, which target folate receptor overexpressing tumor cells. Self-assembled nanoparticles with a hydrodynamic size of 50-200 nm were prepared from poly-γ-glutamic acid and chitosan biopolymers with Gd-ions. The nanoparticles are biocompatible, non-toxic and stable for several months in aqueous media. In vitro assays using confocal microscopy, flow cytometry and MR imaging on HeLa human cervix carcinoma tumor cells showed that folic acid targeted nanoparticles were internalized specifically in a folate receptor dependent manner. In vivo study confirmed, that, considerable accumulation of nanosystems was found compared with the control animal represented by the MR images. Relaxometry measurements demonstrated that the nanoparticle-Gd complexes drastically change the signal intensity of the tumor cells. Because of the contrast enhancement, they are attractive candidates as potential contrast agents for a variety of diagnostic applications including early diagnosis of tumors.

Multispecies animal investigation on biodistribution, pharmacokinetics and toxicity of 177Lu-EDTMP, a potential bone pain palliation agent.

INTRODUCTION: Radionuclide therapy (RNT) is an effective method for bone pain palliation in patients suffering from bone metastasis. Due to the long half-life, easy production and relatively low beta- energy, (177)Lu [T(1/2)=6.73 days, E(beta max)=497 keV, E(gamma)=113 keV (6.4%), 208 keV (11%)]-based radiopharmaceuticals offer logistical advantage for wider use. This paper reports the results of a multispecies biodistribution and toxicity studies of (177)Lu-EDTMP to collect preclinical data for starting human clinical trials. METHODS: (177)Lu-EDTMP with radiochemical purity greater than 99% was formulated by using a lyophilized kit of EDTMP (35 mg of EDTMP, 5.72 g of CaO and 14.1 mg of NaOH). Biodistribution studies were conducted in mice and rabbits. Small animal imaging was performed using NanoSPECT/CT (Mediso, Ltd., Hungary) and digital autoradiography. Gamma camera imaging was done in rabbits and dogs. Four levels of activity (9.25 through 37 MBq/kg body weight) of (177)Lu-EDTMP were injected in four groups of three dogs each to study the toxicological effects. RESULTS: (177)Lu-EDTMP accumulated almost exclusively in the skeletal system (peak ca. 41% of the injected activity in bone with terminal elimination half-life of 2130 and 1870 h in mice and rabbits, respectively) with a peak uptake during 1-3 h. Excretion of the radiopharmaceutical was through the urinary system. Imaging studies showed that all species (mouse, rat, rabbit and dog) take up the compound in regions of remodeling bone, while kidney retention is not visible after 1 day postinjection (pi). In dogs, the highest applied activity (37 MBq/kg body weight) led to a moderate decrease in platelet concentration (mean, 160 g/L) at 1 week pi with no toxicity. CONCLUSION: The protracted effective half-life of (177)Lu-EDTMP in bone supports that modifying the EDTMP molecule by introducing (177)Lu does not alter its biological behaviour as a specific bone-seeking tracer. Species-specific pharmacokinetic behavior differences were observed. Toxicity studies in dogs did not show any biological adverse effects. The studies demonstrate that (177)Lu-EDTMP is a promising radiopharmaceutical that can be further evaluated for establishing as a radiopharmaceutical for human use.

Comparison of the tumorigenic potential of liver and kidney tumors induced by N-nitrosodimethylamine.

The aim of the study was to determine the tumorigenic potential of two cell lines established from N-nitrosodimethylamine induced rat hepatocarcinoma (HeDe) and mesenchymal renal tumors (NeDe). The basis of the distinction is that human cancers are known to overexpress facilitative GLUT transporters and TGF-beta1 protein. These proteins are linked to the increased metabolic energy consumption indicating uncontrolled growth and proliferation. We have assayed not only the expression of GLUT-1, GLUT-3 and TGF-beta1 proteins, but also the uptake of 2-fluoro-[18F]-2-deoxy-D-glucose (18FDG), a tracer for cancer diagnosis. Western blot analysis and whole body autoradiography were used to measure the 18FDG uptake of tumor cells. Elevated 18FDG uptake was measured in both tumor cell lines. Whole body autoradiography provided evidence that the uptake of 18FDG was lower in the necrotic inner part than in the more vascularized outer parts of primary hepatocarcinoma and mesenchymal renal tumors. GLUT-1 overexpression in hepatocarcinoma tumor, and high levels of GLUT-3 were found in the NeDe cell line and in the mesenchymal renal tumor. TGF-beta-1 was overexpressed in hepatocarcinoma and mesenchymal renal tumors. In vitro and in vivo parameters support the view that the tumorigenic potential of cancer cells cannot be determined by the expression of a single parameter such as the expression of either GLUT-1, GLUT-3 or 18FDG uptake. Besides the tumorigenic potential of the hepatocarcinoma, the high metabolic activity of the renal tumor indicated by its 18FDG uptake, GLUT-3 and TGF-beta1 expression, the mesenchymal renal tumor induced by N-nitroso-dimethylamine is not a benign, but an an aggressive renal carcinoma.

Lymphatic spread of mesenchymal renal tumor to metastatic parathymic lymph nodes in rat.

Rat mesenchymal renal tumor cells (NeDe) transplanted under the kidney capsule of F344 rats resulted in metastases in the parathymic lymph nodes. Tumor cells were isolated from these tumor-bearing lymph nodes and 106 cells were implanted under the kidney capsule. Tumor growth after this implantation could be traced within six days. India ink was implanted to prove that there is a connection between the lymphatic vessels of the kidney capsule and the parathymic lymph nodes. The distribution of the radioligand 18FDG in different organs also provided evidence that the parathymic lymph nodes are the primary sites of metastatic tumor growth. Tumor growth was followed after staining sections of biopsies of normal, tumorous kidneys and parathymic lymph nodes embedded in paraffin. The progression of tumor formation was seen as a frontline between the healthy and tumor bearing tissue. This demarcation line was sharp at the beginning of the invasion and at the peripheral regions of the tumor, while the central region infiltrated into the healthy kidney tissue. The initial invasion gradually turned to an infiltration resulting in the disruption of the renal tissue, especially at the periphery. Accumulation of lipids and flow of blood to the lymphatic vessels was due to the lack of angiogenesis, leading to an increased pressure of the interstitial fluid. Interstitial damage ultimately led to the appearance of blood and the growth of tumor cells in parathymic lymph nodes. The kidney capsule-parathymic lymph node complex is proposed as a suitable metastatic model for the isolated in vivo examination of tumor development and for the analysis of secondary tumors.

Daunorubicin and doxorubicin inhibit the [(11)C]choline accumulation in cancer cells.

We studied how very short (10-40min) incubation with anthracycline derivatives modifies the accumulation of PET tumor-diagnostic radiotracers in cancer cells. The human ovarian A2780 and A2780AD, human B lymphoid JY, human epidermoid KB-3-1 and KB-V-1, and smooth muscle DDT1 MF-2 cells were pre-incubated with daunorubicin and doxorubicin, and the uptake of [(18)F]FDG and [(11)C]choline was measured. Anthracycline treatment decreased remarkably the [(11)C]choline accumulation in a concentration dependent manner, while it did not modify significantly the [(18)F]FDG uptake of the cells.

Renal capsule-parathymic lymph node complex: a new in vivo metastatic model in rats.

BACKGROUND: The ultimate cause of cancer death is, in most cases, the appearance of metastases. The aim of the present study was to contribute to animal experimental investigations of metastatic tumor development. MATERIALS AND METHODS: Rat hepatocarcinoma (He/De), mesoblastic nephroma (Ne/De) cells, and in other cases tumor-bearing lymph nodes were transplanted under the renal capsule of F344 rats. Metastatic potential of tumor cells was examined by whole body autoradiography and phosphor image analysis. The organ distribution of cells was also investigated. RESULTS: Transplanted tumor cells resulted in metastases in the parathymic lymph nodes. Implanted India ink also demonstrated connection between the lymphatic vessels of the renal capsule and the parathymic lymph nodes. The metastatic potential was independent of the primary tumor growth rate. CONCLUSION: The renal capsule-parathymic lymph node complex seems to be suitable for the isolated in vivo examination of metastatic development and for the detailed analysis of secondary tumors.

Functional implications of membrane modification with semenogelins for inhibition of sperm motility in humans.

Semenogelin I and II (Sgs) are the major component of human semen coagulum. The protein is rapidly cleaved after ejaculation by a prostate-specific antigen, resulting in liquefaction of the semen coagulum and the progressive release of motile spermatozoa. Sgs inhibit human sperm motility; however, there is currently no information on its effect on the sperm membrane. This study investigated the role of Sgs on human sperm motility through regulation of membrane potential and membrane permeability. Fresh semen samples were obtained from normozoospermic volunteers, and studies were conducted using motile cells selected using the swim-up method. Sgs changed the characteristics of sperm motion from circular to straightforward as evaluated by a computer-assisted motility analyzer, and all parameters were decreased more than 2.5 mg/mL. The results demonstrate that Sgs treatment immediately hyperpolarized the membrane potential of swim-up-selected sperm, changed the membrane structure, and time-dependently increased membrane permeability, as determined through flow cytometric analysis. The biphasic effects of Sgs were time- and dose-dependent and partially reversible. In addition, a monoclonal antibody against Sgs showed positive binding to cell membrane proteins in fixed cells, observed with confocal fluorescence microscopy. These results demonstrate that Sgs modifies the membrane structure, indirectly inhibiting motility, and provides suggestions for a therapy for male infertility through selection of a functional sperm population using Sgs.

Pallidal deep brain stimulation and L-dopa effect on PET motor activation in advanced Parkinson's disease.

BACKGROUND AND PURPOSE The antiakinetic effect of internal Globus pallidus deep brain stimulation (Gpi-DBS) in Parkinson's disease is not clear and not either how this effect is modulated by L-dopa. METHODS Left Gpi-DBS and/or L-dopa effect was studied with auditory paced right-handed sequential movements on (15)O-butanol positron emission tomography (PET) in five patients. Rest and for conditions during movements (DBS off/L-dopa off; DBS on/L-dopa off; DBS off/L-dopa on; DBS on/L-dopa on) were compared with statistical parametric mapping. RESULTS Gpi-DBS activated the right supplementary motor area/premotor (SMA/PMC), and right insular cortex (IC), and as L-dopa decreased the left sensorimotor cortex (M1/S1) activity. L-dopa increased the left ventrolateral thalamus (VLTH), and decreased the left superior parietal cortex (PC) activity. Gpi-DBS and L-dopa interaction showed right SMA/PMC, IC, and left PC activation, decrease of left VLTH, PMC, and dorsolateral prefrontal cortex (PFC) activity. CONCLUSIONS The improvement of bradykinesia with Gpi-DBS is secondary and contributed to the regress of M1/S1-related rigidity and compensatory SMA/PMC, and IC activation. L-dopa and Gpi-DBS alone each reduces M1/S1 overactivity. Interaction ignores this effect, moreover has akinetic effect in the left VLTH, PMC, and PFC. Motor improvement possibly related to left PC and compensatory right SMA/PMC, and IC activation.

Serial noninvasive in vivo positron emission tomographic tracking of percutaneously intramyocardially injected autologous porcine mesenchymal stem cells modified for transgene reporter gene expression.

BACKGROUND: Porcine bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with a lentiviral vector for transgene expression of the trifusion protein renilla luciferase, red fluorescent protein and herpes simplex truncated thymidine kinase (LV-RL-RFP-tTK; positron emission tomography [PET] reporter gene) for in vivo noninvasive tracking of the intramyocardially delivered MSC fate. METHODS AND RESULTS: A closed-chest, reperfused myocardial infarction was created in farm pigs. Sixteen days after myocardial infarction, LV-RL-RFP-tTK-MSCs were injected intramyocardially using electromechanical mapping guidance in the infarct border zone (n=7). PET-computed tomographic metabolic and perfusion imaging was performed after an intravenous injection of 10 mCi [18F]-FHBG and 13N-ammonia PET at 30+/-2 hours and 7 days after LV-RL-RFP-tTK-MSC treatment. Fusion imaging of the [18F]-FHBG PET-computed tomography with MRI was used to determine the myocardial location of the injected LV-RL-RFP-tTK-MSCs. Seven days after injections, [18F]-FHBG PET showed a decreased cardiac uptake with a mild increased pericardial and pleura uptake in the treated animals, which was confirmed by the measurement of luciferase activity. At 10 days, infarct size by MRI in the LV-RL-RFP-tTK-MSC-treated animals was smaller than controls (n=7) (23.3+/-1.5% versus 30.2+/-3.5%, P<0.005). The presence of the LV-RL-RFP-tTK-MSCs (5.8+/-1.1% of the injected cells) in the myocardium 10 days after intramyocardial delivery was confirmed histologically. CONCLUSIONS: Reporter gene imaging enables the tracking of the persistence of viable LV-RL-RFP-tTK-MSC in the peri-infarcted porcine myocardium at 10 days after delivery using clinical PET scanners.

Nutrition and immune system: certain fatty acids differently modify membrane composition and consequently kinetics of KV1.3 channels of human peripheral lymphocytes.

Potassium (K(+)) channels of human peripheral lymphocytes play a considerable role in the signalling processes required for immune responses. Modification of the fatty acid composition of the membrane influences the functions of various membrane enzymes and ion channels. We set out to establish how the incorporation of fatty acids with different carbon chain lengths and degrees of unsaturation into the cell membrane influences the function of K(V)1.3 channels of lymphocytes, thereby potentially modifying the immune responses of the cells. The incorporation of the fatty acids into the cell membrane was monitored by gas chromatography. Whole-cell patch-clamp experiments demonstrated that the polyunsaturated linoleic acid, arachidonic acid and docosahexaenoic acid all decreased the activation and inactivation time constants of the K(V)1.3 channels, but did not affect the voltage-dependence of steady-state activation and steady-state inactivation of the channels. Treatment with the saturated palmitic acid, stearic acid and the monounsaturated oleic acid did not result in significant changes in the biophysical parameters of K(V)1.3 gating studied. We conclude that the incorporation of fatty acids unsaturated to different degrees into the cell membrane of lymphocytes influenced the rate of gating transitions but not the equilibrium distribution of the channels between different states. This effect depended on the degree of unsaturation and the chain length of the fatty acids: no effects of saturated and monounsaturated fatty acids (16:0, 18:0 and 18:1) were observed whereas treatment with polyunsaturated fatty acids (18:2, 20:4 and 22:6) resulted in significant changes in the channel kinetics.

Complete inhibition of P-glycoprotein by simultaneous treatment with a distinct class of modulators and the UIC2 monoclonal antibody.

P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 approximately 20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp+ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.