DNA fragmentation and caspase-independent programmed cell death by modulated electrohyperthermia.

BACKGROUND AND PURPOSE: The electric field and the concomitant heat (electrohyperthermia) can synergistically induce cell death in tumor tissue, due to elevated glycolysis, ion concentration, and permittivity in malignant compared with nonmalignant tissues. Here we studied the mechanism and time course of tumor destruction caused by electrohyperthermia. MATERIAL AND METHODS: Bilateral implants of HT29 colorectal cancer in the femoral regions of Balb/c (nu/nu) mice were treated with a single 30-min shot of modulated, 13.56-MHz, radiofrequency-generated electrohyperthermia (mEHT). Tumors at 0, 1, 4, 8, 14, 24, 48, and 72 h posttreatment were studied for morphology, DNA fragmentation, and cell death response-related protein expression using tissue microarrays, immunohistochemistry, Western immunoblots, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. RESULTS: Modulated EHT treatment induced significant tumor destruction in HT29 xenografts with a peak of a sevenfold increase compared with the untreated controls. The significant treatment-related elevation of DNA fragmentation--detected with TUNEL assay--and apoptotic bodies between 24 and 72 h posttreatment was proof of a programmed cell death response. This was associated with significant mitochondrial accumulation of bax and mitochondrial-to-cytoplasmic release of cytochrome c proteins between 8 and 14 h. Cleaved caspase-3 levels were low and mainly localized to inflammatory cells. The substantial cytoplasmic-to-nuclear translocation of apoptosis-inducing factor (AIF) and its 57-kDa activated fragment detected between 14 and 24 h after treatment indicated AIF as an effector for DNA fragmentation. CONCLUSION: Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Expression pattern of markers in the canine ovarian cycle.

Although hormonal changes during different phases of the oestrous cycle of bitches are well-described, knowledge about the luteal phase and anoestrus is incomplete. Furthermore, which paracrine and autocrine critical factors that differentiate between follicles destined for atresia and those that continue to develop are unknown. In this study, ovarian tissue was collected from 39 healthy bitches that were subject to ovariectomy or ovariohysterectomy for surgical neutering or medical purposes such as unwanted pregnancy. Bitches were allocated to different groups depending on the stage of the oestrous cycle. Serum progesterone, LH, FSH and 17beta-estradiol (E(2)) -levels were determined and immunhistochemistry was performed for a variety of receptor antigens; Ki-67, vimentin, pan cytokeratin antibody, p53 and oestrogen receptor (ER) alpha antigens. Marked differences were found in progesterone concentration between pregnant and non-pregnant animals. Oestrogen concentration was significantly lower in pro-oestrus and ovulation than during the luteal phase. Although progesterone could be detected in cytoplasm of ovarian cells at each stage, its presence was restricted to follicular cells during anoestrus. A strong presence of AE1/AE3, vimentin and p53 was found in each oestrous stage, in contrast with Ki67. The localization of ERalpha appeared to vary during the oestrous cycle, a phenomenon that suggests a switch between target cells of oestrogen; while as a proliferation marker, the mild reaction of p53 during parturition suggests an apoptotic process at this stage of the cycle.

Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats.

AIMS: The altered gut-brain interaction can be in the background of functional gastrointestinal (GI) disorders. In the GI tract, the slow-wave myoelectric signals can be detected by electromyography (EMG). The aims of our study were to follow up the stress induced alteration in the GI tract by smooth muscle EMG in wakeful rats. MAIN METHODS: The GI tract myoelectric activity of male rats was measured by an electrode pair under the abdominal skin, the responses were detected and analyzed by a software using fast Fourier transformation. Animals were immobilized and treated with either diazepam or haloperidol. The plasma corticosterone level was determined by ELISA kit, the levels of drugs were measured by HPLC, while the direct GI effects of the compounds were tested in an organ bath. KEY FINDINGS: Significant correlation (r2 = 0.52) was found between the immobilization induced increase in the EMG spectra of the GI tract segments and the increase in corticosterone plasma levels. The stress-reducing effects of diazepam and haloperidol were also detectable by smooth muscle EMG in the GI tract. No direct smooth muscle actions of the drugs were found in organ bath studies. SIGNIFICANCE: The smooth muscle EMG instrument can measure the level of acute stress and is applicable for the investigation of central nervous system affecting drugs through the GI tract in awake rats. This is the first tool to measure the stress response via the GI tract reactions. The technique may open a new perspective in the diagnosis and therapy of psychosomatic disorders.

Follicular development and plasma concentrations of LH and prolactin in anestrous female dogs treated with the dopamine agonist cabergoline.

The effect of a daily administration of a dopamine agonist (cabergoline, 5 microg/kg) for 4 weeks, starting about 95 days after the end of estrus on follicular development and its relationship with LH and prolactin secretion has been investigated in two groups of anestrous bitches (Beagles and Greyhounds). Pro-estrus was detected in 80% (8/10) of beagles and 50% (3/6) of treated greyhounds. The mean inter-estrus interval of treated animals was 132+/-5.0 and 169+/-7.0 days for beagles and greyhounds, respectively, and in both this differed significantly from the cycle preceding treatment (192+/-9.0 and 198+/-12.0 days) and from that in untreated bitches (194+/-11.0 and 196+/-11.0 days for beagles and greyhounds, respectively (all comparisons at P<0.001). The interval from the beginning of treatment to pro-estrus in responding animals was 13.3+/-1.90 days in beagles and 20.3+/-1.70 days in greyhounds. Cabergoline increased (P<0.001) the length of pro-estrus (10.6+/-0.50 and 11.7+/-0.50 days) in the treated estrus cycle compared to the previous estrus cycle (8.4+/-0.30 and 8.8+/-0.40 days for in beagles and greyhound, respectively). Ovarian enlargement and follicle development was detected by ultrasound in 90% of treated beagles and in 83% of greyhound between the second and third weeks of treatment, but only 80% of beagles and 66% of treated greyhound displayed pro-estrus and estrus. In the treated bitches, mean plasma LH increased (P<0.001) before pro-estrus. There was high variability in mean plasma prolactin levels between animals. These data indicate that the administration of the dopamine agonist cabergoline to anestrous bitches increases mean LH plasma levels and induces follicular development shortly before pro-estrus but this activity is not always followed by pro-estrus and estrus. Finally, prolactin per se does not have a prominent role in the control of folliculogenesis in the bitch.

Immunohistochemical detection of progesterone and cellular proliferation in canine mammary tumours.

Immunohistochemical expression of progesterone and the cellular proliferation marker Ki-67 was assessed in formalin-fixed, paraffin wax-embedded samples from 43 canine mammary tumours. Benign tumours showed high surface membrane progesterone expression (mean 196.42+/-25.91 positively labelled cells per 500 tumour cells) and low cellular proliferation (52.14+/-16.73 positively labelled cells per 500 tumour cells), whilst malignant tumours had low progesterone expression (68.19+/-17.53 positively labelled cells per 500 tumour cells) and higher cellular proliferation (141.72+/-23.65 positively labelled cells per 500 tumour cells), the difference being statistically significant (P<0.005) in both cases. These findings suggest that the majority of progesterone receptors in canine mammary tumour tissue are not associated with bound progesterone. The progression towards malignancy in spontaneously arising canine mammary tumours therefore appears to be associated with a decrease in steroid hormone dependency. Progesterone expression was also noted in the cytoplasm of tumour cells where it may be associated with a cellular repair mechanism. This hypothesis is supported by the finding of significantly higher progesterone content in the cytoplasm of benign tumour cells.

Development of the independent function of fetal thyroid glands in the dog in connection with iodothyronine concentrations in pregnant bitches, fetal fluids, and fetal serum.

Thyroxine (T4) and triiodothyronine (T3) concentrations in pregnant and nonpregnant bitches were measured. The allantoic and amniotic fluid samples were collected separately in the third week of pregnancy, and fetal blood samples were collected in the fourth week of pregnancy. There was no difference between T4 results in the pregnant and nonpregnant animals, but the measured serum concentrations exceeded the healthy range for normal adults. Serum T4 concentrations were lower in the fetus than those in adults (P < 0.01). Fetal T4 concentrations continuously increased and reached 13.38 ± 6.19 nmol/L before birth. The fetal serum T4 concentrations were lower than the T4 concentrations in allantoic and amniotic fluid until the seventh week, and the fetal serum T3 concentrations were lower than those in fetal fluids throughout the pregnancy (P < 0.01). Maximum T3 concentrations in allantoic and amniotic fluid exceeded the concentrations in the fetal and maternal serum. It is conceivable that the considerable differences between maternal and fetal serum T4 concentrations in healthy animals are explained by the T4 impermeability of the placenta. Extremely high maternal T4 (193.5 nmol/L) in 1 bitch was associated with T4 concentrations under the detection limit in the fetal fluids and serum suggesting an inhibitory effect. The T4 concentrations in all the fetal fluids and serum were under the detectable concentration that can be defined by 3.0 nmol/L in that bitch. We have demonstrated that fetal thyroid glands start functioning independently at the same time as thyroid cell formation in the dog, but the overproduction of maternal T4 may have a suppressive effect on fetal iodothyronine production.

Is there a relationship between increased aortic stiffness and segmental left ventricular deformation in elite athletes? (Insights from the MAGYAR-Sport Study).

Introduction Myocardial contractility of the left ventricle (LV) is related to arterial distensibility. Sport activity is frequently associated with changes in both LV and arterial functions. This study aimed to find correlations between three-dimensional speckle-tracking echocardiography-derived segmental LV deformation parameters and echocardiographically assessed aortic stiffness index (ASI) in athletes. This study comprised 26 young elite athletes (mean age: 26.7 ± 8.4 years, nine men). Results Among segmental circumferential strains (CSs), only that of apical anterior (r = 0.40, p = 0.05), septal (r = 0.47, p = 0.01), inferior (r = 0.59, p = 0.001), lateral (r = 0.44, p < 0.05), and midventricular anteroseptal (r = 0.44, p < 0.05) segments correlated with ASI, whereas LV-CS of the midventricular anterior segment showed a correlation tendency. Only longitudinal strain of basal anteroseptal (r = -0.46, p < 0.05) and inferoseptal (r = -0.57, p < 0.01) segments showed correlations with ASI, whereas that of the basal anterior segment had only a tendency to correlate. Some segmental multidirectional strains also correlated with ASI. Conclusions Correlations could be demonstrated between increased aortic stiffness and circular function of the apical and midventricular LV fibers and longitudinal motion of the basal septum and LV anterior wall (part of LV outflow tract) in maintaining circulation in the elite athletes.

Silica@zirconia@poly(malic acid) nanoparticles: promising nanocarriers for theranostic applications.

Silica@zirconia@poly(malic acid) nanocarriers of 110 nm mean diameter were designed, synthesized and characterized for the targeted delivery of diagnostic and therapeutic 99mTc to folate-overexpressing tumors. An important achievement was that a multifunctional l-(-)-malic-acid-based copolymer was formed in situ at the surface of the inorganic cores in a single synthetic step incorporating l-(-)-malic acid, ß-cyclodextrin rings, folic acid moieties, and polyethylene glycol chains. Morphological and in-depth structural analysis of the particles proved their core@shell structure. Stability experiments in aqueous media evidenced that stable suspensions can be obtained from the lyophilized powder in 10 mM phosphate buffer at pH 7.4. During 14-day degradation experiments, the nanoparticles were found to be slowly dissolving (including inorganic core) in saline and also in total cell medium. An in vitro toxicity assay on hepatocytes showed a concentration-dependent decrease of cell viability down to 63 ± 1% at the highest applied concentration (0.5 mg ml-1). Proof of concept experiments of technetium-99m radiolabelling and in vivo labelling stability are presented.

Inherently fluorescent and porous zirconia colloids: preparation, characterization and drug adsorption studies.

Porous, fluorescent zirconia particles of nearly 380 nm diameter were prepared without template molecules or labeling dyes. The porous structure is the result of aggregation-induced particle formation. The inherent fluorescence is assigned to coordinatively unsaturated Zr4+ ions at the sol-gel derived ZrO2 surface. After physico-chemical characterization of the native zirconia particles carboxyl and/or amine bearing drug molecules (d,l-α-difluoromethylornithine - DFMO, ursolic acid - UA and doxorubicin - DOX) were adsorbed onto their surface, and the products were analyzed with Fourier-transform infrared spectroscopy (FTIR), thermogravimetry (TG), small-angle X-ray scattering (SAXS), fluorimetry and zeta potential vs. pH measurements. We have found that DOX complexes coordinatively unsaturated Zr4+ ions without dislocating them, while carboxyl-bearing drugs interact with basic surface Zr-OH sites eliminating some of the carbonate species. The adsorption of UA at the zirconia surface shifts considerably the isoelectric point of the surface and thus provides kinetic stability to the particles at physiological pH. An in vivo biodistribution study in two healthy dogs performed by SPECT/CT detection after 99mTc labeling of the nanocarriers has shown the possibility of drug delivery application.

Comparative endocrinology of the insulin-like growth factor-binding protein.

Emerging early in chordate evolution, the IGF-regulatory axis diverged from an insulin-like predecessor into a vertebrate regulatory system specializing in cell growth activation and allied anabolic functions. Essential to the divergence of the IGF and insulin systems was an early presence of soluble IGF-binding proteins (IGFBPs), which bind IGF peptides at much higher affinity than that of the insulin receptor but at comparable affinities to that of the IGF receptor. IGFBPs have no homology with IGF receptors. Instead, IGFBPs are a derived group of proteins within a superfamily of cysteine-rich growth factors, whose members are found throughout the animal taxa. While blocking IGF actions through the insulin receptor is a fundamental role, IGFBPs evolved within the vertebrate line into centralized, 'integrators' of the endocrine growth-regulatory apparatus. IGFBPs have substantial influences on the distribution and bioavailability of IGF peptides in the cellular and physiological environments, but they have a variety of other properties. The six principal mammalian IGFBPs exhibit an array of specialized properties that appear to be derived from a complex evolutionary history (including cell membrane association, interaction with proteins that post-translationally modify them, direct IGF-independent effects on cells, and others) and they are regulated by a diversity of 'outside' factors (e.g. other hormones, metabolic status, stress). Thus, IGFBPs are multifunctional integrators having diverse physiological 'agendas'. Much less is known about IGFBPs and their properties in the other vertebrate taxa. Increasingly, however, it is being recognized that they play equally important endocrine roles, in both conserved and non-conserved ways, when compared with those currently defined in mammals. This review highlights selected 'comparative aspects' in current IGFBP research, in an attempt to view this essential group of endocrine regulators from a wider, biological perspective.

Autosomal recessive acrorenal syndrome.

We describe two sibs with tetraectrodactyly and oligomeganephronic renal hypoplasia. The parents were unaffected. This syndrome of apparently autosomal recessive origin appears to be the first Mendelian form of the acrorenal developmental field defect identified so far.

Simultaneous determination of tacrine and 1-hydroxy-, 2-hydroxy-, and 4-hydroxytacrine in human plasma by high-performance liquid chromatography with fluorescence detection.

An analytical method was developed for the simultaneous reversed-phase (cyano) high-performance liquid chromatographic determination with fluorescence detection of tacrine and 1-hydroxy-, 2-hydroxy-, and 4-hydroxytacrine in human plasma. Alkalinized human plasma samples were extracted with a mixture of chloroform/l-propanol (90:10, v/v). Extraction recoveries generally ranged from 68% to 83% for all four compounds and did not appear to be concentration dependent over the range examined. Calibration curves were constructed over the following ranges: 0.5-30.0 ng/mL for tacrine (THA) and 4-hydroxytacrine (4-OH-THA), 1.0-30.0 ng/mL for 2-hydroxytacrine (2-OH-THA), and 0.925-46.2 ng/mL for 1-hydroxytacrine (1-OH-THA). The intra- and interassay precision (RSD) for the quality control specimens analyzed during validation were < or = 11.8% and < or = 9.28%, respectively. The intra- and interassay accuracy (RE) for the quality control specimens analyzed during validation ranged from 14.1% to -7.5% and 12.1% to -3.33%, respectively, for all four compounds. The limit of quantitation was estimated as the level of the lowest concentration in the calibration curve for each compound based on acceptable interassay precision and accuracy statistics generated at this concentration. The sensitivity of the method was adequate for the determination of THA, 1-OH-THA, 2-OH-THA, and 4-OH-THA following oral administration of Cognex (40 mg single dose) to normal volunteers.

Oncological scintigraphy in dogs with 99mTechnetium MIBI and DMSA(V)--two case reports.

Oncological scintigraphy is described in two dogs with malignant lymphoma and malignant schwannoma, respectively. The distribution of the radiopharmaceuticals could be visualized for the purpose of detection of neoplasia and eventual metastases, and to evaluate the effectivity of therapy. It is concluded that 99mTechnetium MIBI and 99mTc DMSA (V) scintigraphy is useful for the detection of soft tissue malignancy in dogs.

Psychological effects of chronic haemodialysis.

Ninety-four patients, partly on chronic haemodialysis, partly after successful kidney transplantation, were studied. Progress of perceptual performance was followed up by measurements of the reaction time. It is pointed out that accumulation of uraemic toxins causes permanent damage to the central nervous system. The psychological tests revealed behavioural and personality deviations typical of other chronic diseases, the main features being growing anxiety, neuroticitation, sense of inferiority, egocentric attitude, primitive defense reactions, social introversion, distorted image of the ego, etc. Dependence of the patients on the medical or nursing staff and on the dialysing system also favour these symptoms. The uncertain outcome of the illness and the "over-protective" attitude of the close or the more distant milieu also add to the sense of insecurity and uselessness. The need for individual and group psychotherapy is emphasized.

Diagnosis of hyperadrenocorticism in dogs as compared to human diagnostic methods: a review.

Spontaneous hyperadrenocorticism is a common and well-recognised endocrine disorder occurring in dogs. The biochemical diagnosis of hyperadrenocorticism rests on the documentation of excessive glucocorticoid levels or metabolites in urine or blood. The diagnosis of canine hyperadrenocorticism is historically developed from human methods and based on non-dynamic and dynamic tests and visualisation. In some cases the different ranges of endocrine parameters or metabolites necessitate the modification of human protocols. Elevation of the activity of alkaline phosphatase (AP) and its heat-resistant isoenzyme (SIAP) induced by endogenous or exogenous glucocorticoid excess raise the suspicion of hyperadrenocorticism. Cortisol values in morning urine are related to creatinine concentrations to correct for differences in urine concentration. Theoretically, the administration of dexamethasone (DX) at a relatively low dose (0.01 mg/kg of body weight) can inhibit the pituitary secretion of ACTH and, in turn, decrease endogenous cortisol secretion for as long as 24 to 48 h. Therefore, DX administration to dogs with a functioning adrenocortical tumour would not affect the plasma cortisol concentration at any time following its administration. The high-dose dexamethasone suppression test (HDDS) is based on the observation that the function of adrenocortical tumours is independent of pituitary ACTH and they completely suppress ACTH secretion; therefore, regardless of its dose, dexamethasone is never able to suppress cortisol secretion. HDDS can be combined with the measurement of urinary cortisol/creatinine (c/c) ratio from morning urine samples on three consecutive days. In case of non-suppressible urinary c/c ratio one has to speculate on differentiating adrenal tumour (AT) from non-suppressible pituitary-dependent hyperadrenocorticism (PDH) due to a pituitary tumour arising from the intermediate lobe. Radiocholesterol scintigraphy is a less frequently used technique in the diagnosis of canine Cushing's syndrome (CCS); however, it has the same advantages in the localisation and characterisation of adrenocortical diseases as in humans.

Comparison of echocardiography and gated equilibrium radionuclide ventriculography in the measurements of left ventricular systolic function parameters in healthy dogs.

Left ventricular systolic function was assessed in 12 healthy dogs with equilibrium radionuclide ventriculography. The results of the analysis were compared to traditional echocardiographic measurements. Left ventricular internal dimensions and volume were measured at the time of end-systole and end-diastole. Ejection fraction--one of the most informative parameters of cardiac function--was calculated in each animal. Values (e.g. EDD, ESD, EDV, ESV) measured by the scintigraphic method were significantly (Student's t-test, p < 0.05) higher than the data obtained by echocardiography. Ejection fraction (EF) was the only parameter that did not differ significantly when comparing the two imaging techniques. The difference between the results of parallel measurements was in inverse ratio to the size of the heart.

In vitro and in vivo motility studies of 99mTc HM-PAO labelled sperm cells.

The motility of 99mTc HM-PAO radiolabelled sperm cells, labelled as described previously (Balogh et al., 1992) was studied. The active migration of spermatozoa was demonstrated in capillary tubes containing bovine oestrous mucus, using an in vitro motility analyzer. Like nonlabelled sperm cells, the labelled spermatozoa covered a 4-5 cm distance in the capillary tubes during a 10-min run. In vitro motility testing of labelled spermatozoa from different animal species (cattle, rabbit, sheep, horse) did not reveal significant differences. The distribution of spermatozoa within the female genital tract was studied in a previously described animal model (rabbit) and in two new models (sheep, chicken). This method enables the determination and visualization of sperm distribution by a noninvasive technique. The results of in vivo motility tests pave the way for the introduction of a method of unprecedented specificity, which serves for studying the penetrability of the oviduct to spermatozoa.

Thyroid volumetric measurement and quantitative thyroid scintigraphy in dogs.

Thyroid volumetric measurement combined with quantitative 99mTc-per-technetate thyroid scintigraphy was performed in 62 clinical canine patients having suspected thyroid abnormalities. Euthyroid dogs (n = 22) had a total thyroid size of 3.60 +/- 1.36 cm3, the thyroid/salivary gland region of interest (ROI) ratio was 2.01 +/- 0.55, the thyroid/background ROI ratio was 3.86 +/- 0.90, and 20-min thyroid radioactivity uptake was 1.17 +/- 0.71% of the injected dose (I. D.). By Student's unpaired test, thyroid size of the hypothyroid group (n = 36) was not statistically different from that of the euthyroid dogs, but all other quantitative data (e.g., thyroid/salivary gland ROI ratio = 1.08 +/- 0.56, thyroid/background ROI ratio = 2.32 +/- 0.70, and 20-min thyroid radioactivity uptake = 0.34 +/- 0.22% of the I. D.) were significantly (p < 0.001) lower in hypothyroid than in euthyroid dogs. Evaluating the above-listed quantitative data of separated thyroid lobes by Student's paired test, there was no significant difference between the left and the right lobe either in the euthyroid or in the hypothyroid group.

Clinical renal allograft transplantation in a Dalmatian dog: case report.

A case of successful renal allograft transplantation performed in a two-year-old female Dalmatian dog suffering from end-stage chronic renal failure is reported. A one-year-old male German shepherd with severely injured spinal cord was used as kidney donor. Simultaneous kidney allograft transplantation combined with hypothermic initial perfusion as graft conservation was done, placing the donor kidney into the right iliac fossa of the recipient. The immunosuppression protocol consisted of prednisolone and azathioprine. Regular physical, laboratory, ultrasonographic and scintigraphic examinations were used for assessing both the morphology and the function of the allograft. After a two-week period of hospitalisation the patient was discharged in a remarkably improving condition. The recipient died on postoperative day 45 of respiratory insufficiency resulting from secondary pneumonia and pulmonary oedema. Neither macroscopic nor microscopic abnormalities of the allograft were revealed by necropsy.

Principles of renal transplantation in the dog: A review.

The practically relevant body of knowledge available on renal transplantation in the dog is reviewed. A review of the history of renal transplantation is followed by a detailed description of the immunological fundamentals of transplantation, with especial regard to the structures directing rejection, the significance of D-LA-typing and the MLR test serving for their determination, and the possible methods of nonspecific and specific immunosuppression. The most important criteria of recipient and donor selection are presented, together with the main aspects and medicinal protocol of prednisolone-azathioprine and prednisolone-cyclosporin A immunosuppression within the framework of preparation for the surgical intervention. Among the surgical aspects of renal transplantation, first the recommended anaesthetic techniques are outlined, then the three stages of the surgical procedure, i.e. donor nephrectomy, renal perfusion and renal grafting, are described in detail. The last chapter of the article presents the physical, laboratory and instrumental diagnostic methods of rejection diagnosis, with special regard to their practical importance and different roles played in signalling the rejection.