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.

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.

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.

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.

Na+/Ca2+ exchanger inhibitors modify the accumulation of tumor-diagnostic PET tracers in cancer cells.

AIM: To establish the effects of Na(+)/Ca(2+) exchanger (NCX) blockers on 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) and (11)C-choline accumulation in different cancer cells. METHODS: The tumor cells were incubated with NCX inhibitors, and the uptakes of (18)FDG and (11)C-choline were measured. Flow cytometric measurements of intracellular Ca(2+) and Na(+) concentrations were carried out. The presence of the NCX antigen in the cancer cells was proved by Western blotting, flow cytometry and confocal laser scanning microscopy. RESULTS: The NCX is expressed at a noteworthy level in the cytosol and on the cytoplasmic membrane of the examined cells. Incubation of the cells with three chemically unrelated NCX blockers (bepridil, KB-R7943 or 3',4'-dichlorobenzamil hydrochloride) resulted in an increase in the intracellular Ca(2+) concentration, with a simultaneous decrease in the intracellular Na(+) concentration. The treatment with the NCX inhibitors increased the energy consumption of the tumor cells by 50-100%. Thapsigargin abolished the NCX-induced (18)FDG accumulation in the cells. The NCX blockers applied decreased the (11)C-choline accumulation of all the investigated cancer cells by 60-80% relative to the control. CONCLUSION: A possible masking effect of NCX medication must be taken into consideration during the diagnostic interpretation of PET scans.

Converging evidences on language impairment in Landau-Kleffner Syndrome revealed by behavioral and brain activity measures: a case study.

OBJECTIVE: To assess the linguistic abilities of a boy having Landau-Kleffner Syndrome, and relate the focal disturbance of brain activity due to epilepsy to the cognitive and linguistic deficits. METHODS: Several kinds of assessments were carried out, including epileptic source analysis using electronic source localization methods and PET, neuropsychological assessment of cognitive functions, and assessment of speech perception skills (discrimination of phonetic and stress cues) using ERPs. RESULTS: The source of epileptic activity was localized in the left superior temporal lobe. The neuropsychological assessment showed dissociation between verbal and nonverbal functions, and the performance in former was bellow the normal range. ERPs obtained to the processing of phonetic and stress speech cues indicated that the two cues were processed asymmetrically: the mismatch negativity component (MMN) was obtained for the phoneme difference, but not for the stress pattern difference. CONCLUSIONS: Our data converged as it showed that the patient presented a selective impairment of the language system, and the verbal working memory system appeared to be especially defective. It is suggested that the language deficit is at least partly due to the focal disturbance of those neural networks that underlie the functioning of the working memory system. SIGNIFICANCE: LKS is a childhood language disorder that might serve as a model in studying what happens to the language system if, in the course of development, the essential neural circuits are severely disturbed.

Effects of vinpocetine on the redistribution of cerebral blood flow and glucose metabolism in chronic ischemic stroke patients: a PET study.

The pharmacological effects of the neuroprotective drug vinpocetine, administered intravenously in a 14-day long treatment regime, on the cerebral blood flow and cerebral glucose metabolism in chronic ischemic stroke patients (n=13) were studied with positron emission tomography in a double-blind design. The regional and global cerebral metabolic rates of glucose (CMRglc) and cerebral blood flow (CBF) as well as vital physiological parameters, clinical performance scales, and transcranial Doppler parameters were measured before and after the treatment period in patient groups treated with daily intravenous infusion with or without vinpocetine. While the global CMRglc values did not change markedly as a result of the infusion treatment with (n=6) or without (n=7) vinpocetine, the global CBF increased and regional CMRglc and CBF values showed marked changes in several brain structures in both cases, with more accentuated changes when the infusion contained vinpocetine. In the latter case the highest rCBF changes were observed in those structures in which the highest regional uptake of labelled vinpocetine was measured in other PET studies (thalamus and caudate nucleus: increases amounting to 36% and 37%, respectively). The findings indicate that a 2-week long intravenous vinpocetine treatment can contribute effectively to the redistribution of rCBF in chronic ischemic stroke patients. The effects are most pronounced in those brain regions with the highest uptake of the drug.

Effects of miltefosine on membrane permeability and accumulation of [99mTc]-hexakis-2-methoxyisobutyl isonitrile, 2-[18F]fluoro-2-deoxy-D-glucose, daunorubucin and rhodamine123 in multidrug-resistant and sensitive cells.

Miltefosine is a phospholipid analog that exhibits antineoplastic activity against breast cancer metastases, but its mechanism of action remains uncertain. The aim of this study was to investigate the transport mechanism for the removal of miltefosine and [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) from multidrug-resistant cells. The P-glycoprotein pump function, cell viability, and 99mTc-MIBI and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) uptakes were measured in NIH 3T3 (3T3) and NIH 3T3MDR1 G185 (3T3MDR1) mouse fibroblasts and human lymphoid B JY cells. Miltefosine treatment increased the permeability and fluidity of these tumor cells in a concentration-dependent manner. The multidrug-sensitive cells were 3-4 times more sensitive to miltefosine than the multidrug-resistant ones. The extent of 99mTc-MIBI accumulation in the P-glycoprotein-expressing cells increased in the presence of miltefosine, whereas the rhodamine123 and daunorubicin uptakes of the cells did not change significantly. In the 3T3MDR1 cells verapamil reinstated the rhodamine123 and daunorubicin accumulation, but not the 99mTc-MIBI uptake. Cyclosporin A reinstated the uptakes of 99mTc-MIBI, daunorubicin and rhodamine123 by the 3T3MDR1 cells. In a concentration-dependent manner miltefosine decreased the extents of 99mTc-MIBI, rhodamine123, daunorubicin and 18FDG accumulation in the JY and 3T3 cells. Our findings indicate a common transport mechanism for 99mTc-MIBI and miltefosine, which is distinct from that for rhodamine123 and daunorubicin in MDR cells.

Biphasic accumulation kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile in tumour cells and its modulation by lipophilic P-glycoprotein ligands.

AIM: To study the accumulation and washout kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) in MDR positive and MDR negative tumour cells and how this is modified by lipophilic P-glycoprotein ligands. METHODS: The tumour cells were incubated in the presence and absence of the ligands and the uptakes of 99mTc-MIBI, rhodamine 123 and 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) were measured. RESULTS: The accumulation of 99mTc-MIBI in the tumour cells followed biphasic kinetics. Verapamil and cyclosporin A increased the membrane fluidity and significantly enhanced the 99mTc-MIBI uptake of the MDR negative cells, while the rhodamine 123 uptake was not affected. Verapamil significantly increased the uptake of rhodamine 123 and 18FDG but did not modify that of 99mTc-MIBI in the MDR positive cells. Cyclosporin A significantly increased the 18FDG uptake of the MDR positive and negative tumour cells; these effects were ouabain-sensitive. Depolarization of the cytoplasmic membrane, acidification of the extracellular medium and the administration of CCCP decreased the accumulation of 99mTc-MIBI and rhodamine 123 uptake in the tumour cells. CONCLUSIONS: Lipophilic P-glycoprotein ligands modified the biphasic accumulation kinetics of the 99mTc-MIBI uptakes of MDR negative and positive tumour cells in different and complex ways and could therefore mask the P-glycoprotein pump-dependent changes in tracer accumulation.

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.

[PET scan and double-independent pathologic investigations effectively support the detection of occult primary tumors]. / A PET és a kettós, független patológiai leletezés hatékonyan támogatják a rejtett primer tumorok keresését.

Following the failure of conventional diagnostic procedures, whole-body FDG-PET investigations were carried out in 42 metastatic cancer patients to localize occult primary carcinomas. During the clinical follow-up, the presence of malignant tumor was ruled out in 3 cases, and 2 patients originally believed to have carcinoma were confirmed to be suffering from a malignant hematological disease. These false diagnoses were associated with the use of imaging methods only (2 cases) or cytology only (1 case), lack of double, independent pathological investigations (2 cases) or immunophenotyping (2 cases) and the occurrence of an unrecognized rare tumor in a hospital with a small patient turnover (1 case). The discovered 11 occult primaries (4 lung, 3 breast, 2 hypopharynx and 1 base of the tongue carcinomas and 1 non-Hodgkin lymphoma) led to a 28% diagnostic efficacy of PET (11/39 malignant pathological reports). This efficacy is doubled (60%) if PET reveals < or = 5 malignant lesions and the locations of the pathological foci are tumor-specific. We suggest PET investigations in the search for occult primaries following a controlled pathological diagnosis and the failure of conventional diagnostic procedures.

[Positron emission tomography in the investigation of malignant testicular tumors]. / Pozitronemissziós tomográfia a malignus heredaganatok vizsgálatában.

Thirty-three [18F]-FDG and ten [11C]-methionine (altogether 43) PET studies were performed in 37 (24 non-seminoma and 13 seminoma) patients. All results were assessed on the basis of histology (or cytology) or clinical follow-up. PET scan identified metastatic disease in 13 cases while 30 investigations resulted in a negative medical report. There were 3 false-positive cases and no false-negative results were obtained. The false-positive results were likely to occur due to FDG accumulation in benign lesions. There were no false-positive findings with the use of [11C]-methionine. Sensitivity, specificity and accuracy were 100%, 91% and 93%, respectively, using both tracers.

[In vivo investigation of the A2A adenosine receptor distribution using the [11C]-CSC radioligand]. / Az A2A-adenozin-receptor-eloszlás in vivo tanulmányozása [11C]-CSC-radioliganddal.

A 11C labeled selective adenosine A2A antagonist, (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[11C]-methylxanthine [(11C)-CSC] was prepared reacting (E)-8-(3-chlorostyryl)-1,3,-dimethylxanthine and [11C]-methyl iodide. A primary evaluation of [11C]-CSC as a potential tracer for mapping adenosine A2A receptors by positron emission tomography (PET) was also presented. Autoradiographic studies were carried out on Swiss mice. A high level accumulation of radioactivity was observed in the striatum and medulla oblongata in accordance with previous findings on the specific spatial distribution of A2A adenosine receptors. Dynamic PET studies on rabbits showed a fast brain uptake of CSC, reaching a maximum in less than 2 minutes. Competition experiments with the unlabeled ligand proved [11C]-CSC to bind specifically to the appropriate receptor.

[Auditory event related potential and PET scan: a possibility for the comprehensive evaluation of cognition]. / Auditoros eseményhez kötött potenciál és PET: lehetóség a kogníció folyamatának komplex megközelítésére.

The authors studied the parallel use of event related potential (ERP) and brain activation PET in nine healthy volunteers. The presence of P300 wave in the EEG recording was accompanied by a parallel increase in the blood perfusion of the anterior cingulate gyrus. Furthermore, the functional connectivity between cortical and subcortical structures was also present during the task. This method combines the fine temporal-resolution of ERP and spatial-resolution of PET, which allows for the complex examination of fast cognitive processes. This approach may be useful in the investigation of psychiatric disorders.

[Right prefrontal cerebral hemispheric activation by symmetrical carotid sinus baroreceptor stimulation]. / Jobb agyféltekei praefrontalis aktiváció szimmetrikus carotis sinus baroreceptor-ingerlés hatására.

This study was performed to test the hypothesis of greater right hemispheric involvement in the processing of signals related to baroreceptor stimuli. Carotis sinus baroreceptors were stimulated by rhythmically decreasing air pressure in a neck chamber, and as a control the thorax was stimulated in a similar manner. Changes in regional cerebral blood flow (rCBF) were measured by positron emission tomography (PET). Baroreceptor stimulation resulted in rCBF increase in the right anterior-inferior prefrontal cortex (Brodmann areas [BA] 10/44/47) and bilaterally in BA 6/8. The authors conclude that, at least in some stages of baroreceptor information processing, the right hemisphere plays a greater role than the left one.

[Synthesis of radiopharmaceuticals for PET investigations]. / Radiogyógyszerek elóállítása pozitronemissziós tomográfiás vizsgálatokhoz.

The PET radiopharmaceuticals are prepared on the spot in most cases due to the short lifetime of the isotopes used. The first step of this process is the isotope production by small cyclotrons. The synthons made from the isotopes react with the precursor of the given radiopharmaceutical. The target compound selected from the reaction mixture is ready for injection after purification, formulation and sterile filtration. In addition to [18F]-FDG, [11C]-methionine and [15O]-butanol routinely used for diagnostic purposes in the PET Centre of the University of Debrecen, a number of other radiopharmaceuticals are synthesized for use in research from time to time.

[The [18F]-FNECA serves as a suitable radioligand for PET investigation of purinergic receptor expression]. / A [18F]-FNECA széleskörúen alkalmazható radioligand a purinerg receptorexpresszió PET-vizsgálatához.

The well known and widely used P1 adenosine agonist, 5'-N-ethyl-carboxamidoadenosine (NECA), was labelled with 18F isotope for the in vivo PET investigation of A1, A2 and A3 adenosine receptor expression. The precursor 2-[18F]fluoroethylamine was reacted with 2',3'-O-isopropylideneadenosine-5'-uronic acid. Specific activity of the [18F]-FNECA was (2.3 +/- 1.1) TBq/mmol (60 Ci/mmol). Dynamic PET measurements were carried out in rabbits to study the in vivo kinetics of the receptor saturation with the labelled ligand. The time dependent accumulation was followed up in the heart, lungs, liver, brain and testis. The radiotracer uptake was rapid and reached its maximum in less than two minutes in the heart and testes after v. injection of the radiopharmaceutical, while it took about 6 minutes in the brain, lungs and liver. High [18F]-FNECA accumulation was detected in the intestines, too. The specific binding of the [18F]-FNECA was tested in competition experiments in brain and heart sections using autoradiographic technique. The outlined synthesis provided sufficient amounts of [18F]-FNECA to map adenosine receptor expression under physiological conditions.

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.