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.

Comparison of the kidney fungal burden in experimental disseminated candidiasis by species of the Candida parapsilosis complex treated with fluconazole, amphotericin B and caspofungin in a temporarily neutropenic murine model.

BACKGROUND: The efficacy of fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) was tested against three Candida orthopsilosis, three C. metapsilosis and two C. parapsilosis sensu stricto isolates in neutropenic mice. METHODS: Mice were immunosuppressed by 200 mg/kg cyclophosphamide. Five-day intraperitoneal treatment was started 24 h after infection. Kidney burden was analyzed using the Kruskal-Wallis test. RESULTS: FLU 10 and 20 mg/kg as well as AMB 1 mg/kg significantly decreased the fungal burden (p < 0.05) for all eight isolates of the three species. CAS 2 and 5 mg/kg were efficacious against all C. orthopsilosis and C. metapsilosis isolates (p < 0.05), but only 5 mg/kg CAS was effective against C. parapsilosis isolates (p < 0.05). CONCLUSIONS: The efficacy of FLU and AMB against the three species was comparable. Though the activity of CAS was higher against C. orthopsilosis and C. metapsilosis, the current treatment guidelines for C. parapsilosis sensu stricto seem to be applicable to other 'psilosis' species.

Efficacy of a single 6 mg/kg versus two 3 mg/kg caspofungin doses for treatment of disseminated candidiasis caused by Candida albicans in a neutropenic mouse model.

We compared the efficacy of single six mg/kg and 2x3 mg/kg caspofungin doses to the traditional one mg/kg daily against C. albicans in a neutropenic murine model. In lethality experiments, all regimens improved survival (p<0.0014 for all three isolates); differences among the treated groups were not statistically significant. We calculated kidney fungal burdens on each study day for six days postinfection using two isolates in two experiments. In the first three days, only the six mg/kg dose produced significant decrease on all study days (p<0.05-0.001), but differences between the three treatment arms disappeared by 4-6 days postinfection (p<0.05 for all isolates on all days).

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.

Unique morphological alterations of the HTLV-I transformed C8166 cells by infection with HIV-1.

C8166 cells express T lymphocyte markers, a monocyte-specific esterase, taxpolypeptide of HTLV-I. In spite of this transactivator, their HIV-1 yield is low. Their culture conditions were modified, and infected cells were immobilized on a poly-L-lysine sheet under semisolid overlays to study their phenotypic alterations and HIV-1 production by microscopy and electron microscopy. Another lymphoid cultures (MT-4, CEM, CEM-ss, AdCEM) similarly treated were infected with either HIV-1/RF or IIIB. Specificity of HIV-1 was compared to the effects of vesicular stomatitis virus (VSV). Unlike other cultures, HIV-1/RF infected C8166 cells in Eagle s MEM exhibited surface projections resembling hairy leukemia cells, which was followed by balloon degeneration and apoptosis. Immobilized HIV-1 infected cultures formed flat syncytia with several interdigitating dendritic projections. Syncytia shrunk with condensed nuclear material and axon-like filaments characteristic for infected macrophages. VSV induced enlargement and necrotic lysis of all cell types. Early postinfection with HIV-1, electron microscopy revealed irreversible membrane fusion above cell nuclei, and transient fusion between filaments. Transient presence of coated vesicles containing intact HIV-1 particles, Birbeck granule-like structures of Langerhans cells, fibrillar-lamellar structures resembling hairy leukemia or Sézary cells were detected. Late postinfection, high proportion of HIV-1 bud from polarized cytoplasm was empty particle, while that bud and entrapped in cytoplasmic vacuoles contained two or multiple cores in a fused envelope. The effect of early gene products of HIV-1 on HTLV-I and C8166 cells might elicit their latent potentials for monocyte or interdigitating dendritic cells, while in the later phase HTLV-I products might alter HIV-1 virion assembly.

On the use of semi-logarithmic plots for baseflow separation.

Recession flow has long been considered a composite of exponential terms, where each exponential term represents a different source of water discharged from the watershed. The changing slopes in the semi-logarithmic plot of the discharge have been considered indicative of the decreasing contribution of surface runoff and interflow to the discharge. The results of this analysis show that the changing slope in the recession plot can be the consequence of baseflow drainage. This can invalidate the semi-logarithmic baseflow separation technique when applied to some hydrologic settings.

A chronically instrumented rat model to assess the altered baroreflex due to exercise.

We developed a conscious, chronically instrumented, exercise-trained rat model and examined the time course of the training-induced alteration of baroreflex function in response to hypertensive conditions. Exercise-trained (ET) animals ran at 18 m.min-1, 15% grade, for 60 min.d-1, 5 d.wk-1 for 5 wk. Baroreflex tests were conducted on day 6 each week. Regression line slopes relating the change in mean arterial pressure (delta MAP) to the change in heart rate (delta HR) were used to assess baroreflex sensitivity. Intravenous injections of phenylephrine were used to create hypertensive conditions. Compared with the C group, slopes of ET animals were reduced (from 2.1 to 1.2 bpm.mm Hg-1, P < 0.05) as early as week 3 of training in response to increasing doses of PE, and reached 0.8 bpm.mm Hg-1 by the end of training. The reflex bradycardiac response (delta HR) to PE was reduced (P < 0.05) depending on the dose of PE and the duration of training: in micrograms PE.kg-1 body weight, 5 (71% +/- 6% of control at week 2), 3 (70% +/- 7% of control at week 3, and 1 (61% +/- 10% of control at week 4). The pressor (delta MAP) to PE remained constant throughout training. Thus, using a chronically instrumented rat model that maintains the ability to run, we observed that the ability of the arterial baroreflex to produce bradycardia during pressor events was substantially reduced following as few as 2 wk of training.

Herpes simplex virus L particles contain spherical membrane-enclosed inclusion vesicles.

The fine structure of light (L) particles of herpes simplex virus type 1 was examined by cryo-electron microscopy and compared to that of virions. The L particles appeared to be spherical entities with significant variation in size, on average smaller in diameter than virions (140 nm compared to 180 nm). The technique confirmed that L particles are composed of an outer envelope, i.e. a bilaminar membrane with protruding glycoprotein spikes, and a uniformly granular tegument, but lack any nucleocapsid. In addition it revealed the presence of one or occasionally more spherical objects, termed inclusion vesicles (IVs), embedded in the tegument of a large proportion of L particles but not observed in virions, suggesting that presence of IVs is unique to the L particles. The IVs vary in size and appear to be composed of a bilaminar membrane without surface projections and filled with material of relatively low electron density, suggesting that the composition of IVs is distinct from that of the envelope and tegument of the L particles.

Naloxone-induced potentiation of cardiac alpha-2 adrenoceptor-mediated depression of neurogenic tachycardia.

The objective of this investigation was to test the hypothesis that naloxone directly activates alpha-2 adrenoceptors to cause depression of neurogenic tachycardia as suggested in an earlier investigation (Naloxone-Induced Bradycardia in Pithed Rats: Evidence for an Interaction with the Peripheral Sympathetic Nervous System and Alpha-2 Adrenoceptors. J. Pharmacol. Exp. Ther. 296: 916-926, 1992). Bolus doses of naloxone in a range of 10-1000 micrograms/kg i.v., administered in the presence of sustained neurogenic tachycardia (108 +/- 10 beats per min), resulted in a dose-dependent inhibition of neurogenic tachycardia with a maximum inhibitory response of 21% and an ED50 of 55 +/- 2.3 micrograms/kg. The inhibition of the naloxone-induced inhibition of neurogenic tachycardia was antagonized by phentolamine (5 mg/kg i.v.) and rauwolscine (0.5 mg/kg i.v.), but not prazosin (0.1 mg/kg i.v.). In the absence of sympathetic nerve activity, low doses of naloxone (10-300 micrograms/kg i.v.) had no effect on heart rate. These data suggest that naloxone in lower doses (10-1000 micrograms/kg i.v.) is a partial agonist at prejunctional alpha-2 adrenoceptors. In the presence of a steady-state maximum response (21% inhibition of neurogenic tachycardia) caused by naloxone infusion (100 and 1000 micrograms/kg/min i.v.), the ED50 of the preferential alpha-2 adrenoceptor agonist, UK14304-18, was not shifted to the right, but instead shifted to the left. This suggests that naloxone-induced depression of the neurogenic tachycardia does not involve the direct activation of alpha-2 adrenoceptors, but involves the potentiation of alpha-2 adrenoceptor-mediated inhibition of heart rate through an unknown mechanism.

Naloxone-induced bradycardia in pithed rats: evidence for an interaction with the peripheral sympathetic nervous system and alpha-2 adrenoceptors.

Earlier experiments performed in this laboratory have demonstrated that naloxone infusion (1 mg/kg/min i.v.) into conscious rats results in a bradycardia that has a peripheral component, is dependent on a certain level of sympathetic activity and is sensitive to alpha adrenoceptor blockade (5 mg/kg of phentolamine i.v.). The main objective of this investigation was to examine the underlying mechanism(s) responsible for the peripherally mediated naloxone-induced bradycardia, and to test the hypothesis that naloxone interacts with peripheral inhibitory alpha adrenoceptors associated with depression of peripheral sympathetic activity. Naloxone infusion (1 mg/kg/min i.v.) in pithed rats, in the absence of sympathetic nerve activation, resulted in a bradycardia that could not be blocked by 1 mg/kg (i.v.) of atropine, 5 mg/kg (i.v.) of phentolamine, 0.1 mg/kg (i.v.) of prazosin or 0.5 mg/kg (i.v.) of rauwolscine. Isoproterenol or norepinephrine-induced tachycardia was not blocked by naloxone infusion, suggesting that naloxone does not antagonize the postjunctional activation of cardiac adrenoceptors to cause bradycardia. In the presence of sympathetic nerve activity, naloxone depresses neurogenic tachycardia. This effect was blocked completely by 5 mg/kg (i.v.) of phentolamine or 0.5 mg/kg (i.v.) of rauwolscine, but not 0.1 mg/kg (i.v.) of prazosin or 1 mg/kg (i.v.) of atropine. The results of this investigation suggest that the naloxone-induced bradycardia in pithed rats is mediated postjunctionally and prejunctionally, and that this prejunctional effect is dependent on sympathetic nerve activity and inhibitory alpha-2 adrenoceptors. Furthermore, these results confirm results obtained from conscious rats in an earlier investigation.

The influence of cell culture and storage conditions on HIV-1 infectivity and fusogenic activity.

We have previously demonstrated that acidic medium inhibits the replication of HIV-1. The present study was designed to examine the effects of other growth conditions and infection of fibroblasts by coculture with HIV infected lymphoid cells. Several lymphoblastoid cell lines normally grown in RPMI-1640 were grown in Eagle's MEM. These cells supported virus replication to higher titres than did RPMI-1640. Peak viral titres were achieved within 24-48 h after newly infected or chronically infected cells were placed in fresh medium. When virus was stored in liquid medium either frozen or at higher temperatures, virus titres were retained for several months while frozen but decreased upon storage at 4 degrees C or higher. If cells were passaged after trypsinization in Ca(++)-depleted medium, then a decreased susceptibility of cells for HIV-1 by 2 log10 at 24 h post infection was observed. Infectivity of cell-free and cell-associated HIV-1 was measured using syncytium formation, reverse transcriptase activity and p24 antigen. No fusion between HIV-1 infected CD4+ lymphoblasts and CD4- fibroblasts was observed but HIV-1 infected lymphoid cells, even in the absence of syncytium formation, exerted a strong toxic effect on fibroblasts. This study extends previous findings that medium acidity was inhibitory to virus replication and survival. Thus, conditions for study of HIV must be well controlled in buffered medium so that misleading results are not obtained regarding virus multiplication and possibly regarding transmission to and pathogenesis in CD4- cells.

A method for maintaining and protecting chronic arterial and venous catheters in conscious rats.

The ability to monitor arterial blood pressure and heart rate directly, as well as to sample venous blood, or inject pharmaceutical agents intravenously is important in pharmacological studies of the cardiovascular system. The rat is a frequently used and accepted animal model for cardiovascular investigations, especially those relating to hypertension. Even though the rat is a major model for these studies, the size of the rat has made it difficult to maintain catheters for a long period of time. Although there have been previous methods available, the authors report on an improved method to implant, maintain, and protect arterial and venous catheters in conscious rats for extended periods of time. A Silastic/Tygon catheter is implanted intraarterially and intravenously, exteriorized, and protected with a spring device. Catheters remained patent throughout a 5-week period during which time direct blood pressure recordings were obtained and baroreflexes were evaluated in conscious, unrestrained rats. The described design and methods provide an inexpensive means to maintain chronically implanted venous and arterial catheters in the conscious rat. Furthermore, rats may be gang housed.

Identification and characterization of a novel non-infectious herpes simplex virus-related particle.

During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.

Psychosocial stress elevates blood pressure via an opioid dependent mechanism in normotensive rats.

Stress is an important risk factor in cardiovascular diseases, including hypertension. Endogenous opioids are known to be elevated in stress and in various models of hypertension with differing etiologies. Blockade of endogenous opioids with naloxone has been demonstrated to attenuate or reverse the elevation in blood pressure in both renovascular and spontaneous hypertension. In the current study, increased blood pressure was induced using a model of psychosocial stress. During the first week of stress, systolic blood pressure rose rapidly to reach a level that was sustained throughout the remaining period of stress. Chronic infusion of the opiate antagonist, naloxone, both prevented and completely reversed the elevated blood pressure due to psychosocial stress. These data demonstrate that elevated endogenous opioids are important factors in cardiovascular regulation and are likely to influence both the development and maintenance of stress-induced hypertension.

[Different sensitivity to acid reaction of the AIDS virus and virus-producing cells: clinical conclusions]. / A szerzett immunhiány (AIDS) vírusának és a vírustermelö sejteknek eltérö érzékenysége savi vegyhatás iránt: klinikai következtetések.

Sensitivity of the cell-free human immundeficiency virus type 1 (HIV-1) and its producer cells was Studied in acidic media between pH 7.4 and 4.9 vitro. The cytopathic effect, reverse transcriptase activity and p24 antigen production by survived viruses were monitored in indicator cell cultures. It was established that, the cell-free HIV-1 particles are very sensitive to acidity. Between pH 7.4 and 6.0 they loose infectivity gradually, but this process is irreversible under pH 6.0 and subsequent neutralization cannot restore lost infectivity. However, viability, of virus producer cells is hardly affected between pH 7.4 and 4.9, but their ability to release infectious particles is lost gradually, similarly to the case of cell-free viruses. Neutralization of the media after treatment results in gradual restoration of releasing infectious viruses. These data explain that, cell-free HIV-1 looses infectivity in the acidic vagina or does on the skin, but infectivity is preserved in the blood, semen, rectum and breast milk being neutral or slightly alcalic. Virus carrier or producer lymphocytes by any route of infection can survive such protective mechanism of the body.

Endogenous opiates and the pathogenesis of hypertension.

Opiates are now known to be important modulators of cardiovascular function in both the normotensive and hypertensive states. There is accumulating evidence that endogenous opiates are elevated in models of hypertension of various etiologies including genetic and renovascular hypertension. Early evidence for elevated opiates in hypertension arose from observations that hypertensive humans and rats with genetic or experimental hypertension exhibited hypoalgesia in various tests of pain sensitivity. Because pain and cardiovascular regulatory systems have in common a number of brain loci, cardiovascular effects of opiates and opiate blockade were studied. These studies have shown that opiate blockade can attenuate the development of hypertension and reduce blood pressure in chronic hypertension possibly via actions on the baroreflexes and/or by modulating the centrally mediated pressor actions of angiotensin II.

Endogenous opiate modulation of baroreflexes in normotensive and hypertensive rats.

It is evident that hypertension is associated with elevated endogenous opiates. This study was designed to examine the role of endogenous opiates in the development and/or maintenance of two-kidney renovascular hypertension and in baroreceptor reflex function in conscious hypertensive rats. Naloxone administration during the onset of hypertension significantly attenuated the rise in blood pressure. After one week, systolic blood pressure in naloxone-treated rats was 27 mmHg lower than in 0.9% NaCl-treated hypertensive rats. Acute naloxone infusions in chronic hypertensive animals also significantly lowered blood pressure (-10%) and heart rate (-26%). Baroreceptor function was significantly enhanced in both normotensive (+135%) and hypertensive (+207%) rats after administration of naloxone. Furthermore, naloxone treatment also caused the baroreflex response to shift from the higher reset state toward that seen in normotensive counterparts. The inability of naloxone methyl bromide to alter baroreflex sensitivity indicates that the site(s) of action of opiates resides in the brain. These data support a role for opiates in the development and/or maintenance of renovascular hypertension, which may be related to alterations in baroreceptor reflex function.

Regional blood flows and cardiac function changes induced by angiotensin II in conscious dogs.

Hemodynamic properties of angiotensin (ANG) II 1, 5, 10 and 100 ng/kg i.v. and 10, 100 and 1000 ng/kg i.v.t. were assessed in conscious dogs. ANG II i.v. produced a dose-dependent pressor response (59 +/- 5-124 +/- 16 mmHg) and renal vasoconstriction (1.3 +/- 0.4-96 +/- 32 mmHg/ml/min). Ganglionic blockade (chlorisondamine 2 mg/kg i.v.) diminished mean arterial responses without altering peptide effects on renal circulation. At the highest dose, ANG II i.v. induced cardiac stimulation: increased heart rate (75 +/- 4-115 +/- 6 beats/min), cardiac output (2.0 +/- 0.1-2.4 +/- 0.2 l/min), dP/dt (2308 +/- 181-2773 +/- 173 mmHg/sec) and coronary blood flow (49 +/- 10-96 +/- 23 ml/min). Although with chlorisondamine cardiac response was more pronounced, subsequent beta blockade abolished it. Concomitantly, an isolated increase in plasma epinephrine was recorded (63 +/- 8-1505 +/- 354 pg/ml). A pressor response (59 +/- 8-89 +/- 13 mmHg) and renal vasoconstriction (1.1 +/- 0.1-2.2 +/- 0.5 mmHg/ml/min) were also produced by ANG II i.v.t. at the highest dose. These centrally mediated changes were prevented by chlorisondamine. Our study demonstrates 1) i.v. ANG II-mediated pressor responses are dependent on direct and indirect components, the relative contribution of each being dependent on the regional circulation; ANG II i.v. also produced a biphasic cardiac response--an initial centrally mediated depression and a secondary stimulation dependent on epinephrine via cardiac beta receptors and 2) i.v.t. ANG II-mediated pressor effects are essentially indirect. Finally, no evidence was found to support the role of vasopressin in ANG II effects.

Cryoblockade of the ventromedial frontal cortex reverses hypertension in the rat.

The anteroventral part of the hypothalamus adjacent to the third ventricle (AV3V) has been implicated in electrolytic lesion studies as a site crucial to the development and maintenance of hypertension. Cryoblockade is known to alter synaptic and axonal transmission differently at different temperatures. In this study, cooling of the hypothalamus, including the AV3V area, to the temperature known to block only synaptic function did not alter blood pressure in two different models of experimental hypertension in the rat. Cooling sufficient to block both synaptic and axonal transmission, however, reduced blood pressure elevations to near normotensive levels. Synaptic cryoblockade in the ventromedial portion of the frontal cortex lowered experimental hypertension by 21 +/- 3 mm Hg (p less than 0.05). In normotensive controls, blood pressure was not altered by cryoblockade in either the frontal cortex or hypothalamus. Anatomical evidence provided by others shows that cells in the ventromedial frontal cortex project, in part, through the AV3V region to the brainstem cardioregulatory structures. These results indicate that neural activity arising in frontal cortex is axonally projected through the hypothalamus to maintain elevated blood pressure in experimental hypertension.