RESISTANCE OF TRYPANOSOMA-CRUZI TO BLOOD CLEARANCE INDUCED BY ACUTE-PHASE IMMUNE MOUSE SERUM

To investigate functional changes in Trypanosoma cruzi parasites induced during their interaction with the vertebrate host, we compared the blood clearance profiles of blood forms isolated from infected normal mice (Reg-Tc) or from infected mice immunodepressed after treatment with cyclophosphamide (Cy-Tc). Parasite blood numbers were measured at various time intervals in animals injected intravenously (i.v.) with 1-2 x 10(6) T. cruzi of either isolate. In the absence of added immune sera (spontaneous clearance), Reg-Tc and Cy-Tc were cleared from blood at similar rates. However, when acute immune mouse serum (Ac-IMS) was injected i.v. 2 min after inoculation of parasites, a significant proportion of Cy-Tc only was cleared from the blood an hour later, whereas Reg-Tc were not, their clearance profile being identical to that observed in mice injected with normal mouse serum. Cy-Tc susceptibility to Ac-IMS was not the result of a toxic effect of cyclophosphamide over T. cruzi as parasites recovered from animals immunodepressed by irradiation before infection were cleared similarly by acute serum. Contrary to Ac-IMS, chronic immune mouse serum induced similar rates of disappearance of Reg-Tc and Cy-Tc from blood. Our results suggest the occurrence of T. cruzi selection or modification during the acute phase, which leads to an increased parasite resistance to the clearance properties of acute-phase antibodies.

ANTAGONISM OF THE MYOTOXIC EFFECTS OF BOTHROPS-JARARACUSSU VENOM AND BOTHROPSTOXIN BY POLYANIONS

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.

2-DEOXYGLUCOSE-INDUCED FOOD-INTAKE BY NILE TILAPIA, OREOCHROMIS-NILOTICUS (L)

The alimentary and glycemic responses to cytoglycopenia were studied in thirty-one Nile tilapia alevins of indeterminate sex and age, measuring on average 10.67 +/- 0.82 cm. The cytoglycopenia was provoked by ip injection of 60 mg/kg 2-deoxy-D-glucose (2-DG, N = 16). The control group (N = 15) was submitted to ip injection of 0.2 ml saline. Blood samples for glucose determination were obtained before and three hours after drug administration by cardiac puncture. Food was then offered ad libitum. One hour later the animals were sacrificed and their stomachs removed. The difference in wet weight between full and empty stomach was utilized to quantify the food intake. Median food intake was 0.3877 g for the fish treated with 2-DG and 0.107 g for the animals injected with saline. This difference was statistically significant by the Mann-Whitney test (P<0.05). The median values of blood glucose levels before drug injection were 46.19 mg/100 ml in the 2-DG-treated fish and 44.54 mg/100 ml in the control group. Three hours after drug administration, the values were 48.64 mg/100 ml in the experimental group and 56.90 mg/100 ml in the control group. The difference between the values of blood glucose before and after the drug was not significant for either group. We conclude that glucoprivation provokes food intake in fish and that the same glucoprivation was not sufficient to provoke hyperglycemia.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari : Ixodidae)

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Amblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 mug extract in 0.03 rut of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo(R) device before and 10 min, 1, 2,4,18, 24,48,72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60 % increase in ear thickness) and a delayed reaction (18 h) (85 % increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60 % increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

Abnormal expression of bcl-2 and bax in rat tongue mucosa during the development of squamous cell carcinoma induced by 4-nitroquinoline 1-oxide

4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis is a useful model for studying oral squamous cell carcinoma. The aim of this study was to investigate the expression of bcl-2 and bax during tongue carcinogenesis induced by 4NQO. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Although no histological changes were induced in the epithelium after 4 weeks of carcinogen exposure, bcl-2 and bax were over-expressed (P < 0.01) in all layers of the 'normal' epithelium. The expression levels were the same in all layers of epithelium for both the antibodies used (bcl-2 or bax). In dysplastic lesions at 12 weeks following carcinogen administration, the levels of bcl-2 and bax expression did not increase when compared to negative control with the immunoreactivity for bcl-2 being restricted to the superficial layer of epithelium. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, bcl-2 was expressed in some cells of tumour islands. on the other hand, immunostaining for bax was widely observed at the tumour nests. The labelling index for bcl-2 and bax showed an increase (P < 0.05) after only 4 weeks of 4NQO administration. In conclusion, our results suggest that abnormalities in the apoptosis pathways are associated with the development of persistent clones of mutated-epithelial cells in the oral mucosa. Bcl-2 and bax expression appears to be associated with a risk factor in the progression of oral cancer.

EARLY ACTIVATION OF SPLENIC MACROPHAGES BY TUMOR-NECROSIS-FACTOR-ALPHA IS IMPORTANT IN DETERMINING THE OUTCOME OF EXPERIMENTAL HISTOPLASMOSIS IN MICE

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

EFFECT OF ELECTROLYTIC AND CHEMICAL LESION BY IBOTENIC ACID OF THE AMYGDALA ON SALT INTAKE

Sodium chloride intake was studied in male Holtzman rats weighing 250-300 g submitted to electrolytic and chemical lesion of the cell bodies, not fibers of the amygdaloid complex. Sodium chloride (1.5%) intake increased in animals with electrolytic lesion of the corticomedial nucleus of the amygdala. Sodium chloride (1.5%) intake increased after ibotenic acid injection into the corticomedial nucleus of the amygdala to a larger extent (26.6 +/- 9.2 to 147.6 +/- 34.6 ml/5 days). The results indicate that sodium intake response can be induced by lesions, which involved only cell bodies. The fibers of passage of the corticomedial nucleus of the amygdala produce a water intake less consistent than that induced by ibotenic acid, which is more acute. The results show that cell bodies of this region of the amygdala are involved in the control of sodium chloride intake.

Male gonadal denervation by guanethidine at pre-puberty: Different doses, different results. The intervention of the pineal deafferentation

Since gonadal denervation and pineal deafferentation by cervical superior ganglionectomy affect sexual development, this study was performed to evaluate testicular steroidogenesis, spermatogenesis and the cervical superior ganglion (CSG) histology in rats treated with guanethidine (GD). The treatment was performed by GD s.c. injections for 3 weeks, from the 21st day of age to the 41st day of age (pre-puberty), when the animals were sacrificed. Different doses were used: group A=10 mg/kg/day, group B=50 mg/kg/day and saline (control group). Testicular denervation was confirmed by HPLC for catecholamines in testicular tissue. Testicular concentrations (TC) of progesterone (P4) and testosterone (T) were measured by RIA. Significantly higher TC of P4 and lower TC of T were observed only in group A in comparison with group B and the control group. No alteration of sperm production was observed in either treated group. Histological analysis of CSG showed only few neuronal alterations in group A rats, while in group B the nervous cells were practically destroyed. This suggests that 10 mg/kg/day GD treatment probably produces a specific blockade of 17 alpha-hydroxylase/17,20 desmolase at pre-puberty leading to a decrease of the androgen production. However, in the 50 mg/kg/day group no differences were observed concerning the steroid profiles, this result being attributed to the extensive damage to the CSG observed only in group B. The CSG destruction causes deafferentation of the pineal gland producing abolishment of the inhibition of the 17 alpha-hydroxylase/17,20 desmolase promoted by melatonin or by an out of phase production of androgen.

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis.

Properties and in vivo investigation of nanocrystalline hydroxyapatite obtained by mechanical alloying

Mechanical alloying has been used successfully to produce nanocrystalline powders of hydroxyapatite (HA) using three different procedures. The milled HA was studied by X-ray diffraction, Infrared, Raman scattering spectroscopy and Scanning Electron Microscopy (SEM). We obtained HA with different degrees of crystallinity and time of milling. The grain size analysis through SEM and XRD shows particles with dimensions of 36.9, 14.3 and 35.5 nm (for (R1), (R2) and (R3), respectively) forming bigger units with dimensions given by 117.2, 110.8 and 154.4 nm (for (R1), (R2) and (R3), respectively). The Energy-Dispersive Spectroscopy (EDS) analysis showed that an atomic ratio of Ca/P= 1.67, 1.83 and 1.50 for reactions (R1), (R2) and (R3), respectively. These results suggest that the R1 nanocrystalline ceramic is closer to the expected value for the ratio Ca/P for hydroxyapatite, which is 513 congruent to 1.67. The bioactivity analysis shows that all the samples implanted into the rabbits can be considered biocompatible, since they had been considered not toxic, bad not caused inflammation and reject on the part of the organisms of the animals, during the period of implantation. The samples implanted in rabbits had presented new osseous tissue formation with the presence of osteoblasts cells. (C) 2004 Elsevier B.V. All rights reserved.

Differential susceptibility to chromatid breaks induced by bleomycin in sub-fertile and fertile bovines

The rate of chromatid breaks was studied in cows with a history of sub-fertility by means of a test based on measurement of the average of breaks induced in lymphocytes of peripheral blood cultures. Fourteen female specimens were divided into two groups: fertile and sub-fertile. Peripheral blood lymphocytes were cultured and prepared for cytogenetic analysis. Two types of culture were established for each animal to evaluate the response of peripheral blood lymphocyte cultures to the genotoxic effects of bleomycin. The first culture did not receive bleomycin treatment (spontaneous chromosome aberrations). Our results showed that median breaks per cell (b/c) (+/-semirange) for spontaneous culture of the fertile and sub-fertile animals and bleomycin sensitivity assay for fertile and sub-fertile animals were 0.00 +/- 0.06, 0.02 +/- 0.03, 0.08 +/- 0.05 and 0.22 +/- 0.09, respectively. There was no significant difference (P > 0.05) in the chromosomal breakage in lymphocytes not exposed to bleomycin; however, in comparing the number of chromatid breaks per cell in cultures treated with bleomycin, the sub-fertile group showed a significantly higher (P < 0.05) level than the fertile group. These findings have implications both for identifying cattle with less than optimum fertility as well as for providing potential avenues to study the origins of sub-fertility. (C) 2004 Elsevier B.V. All rights reserved.

Effect of intracerebroventricular injection of ramipril on the drinking response caused by injection of noradrenaline into the third ventricle

We studied the effect of ramipril injected into the third ventricle (3rdV) on the control of water intake induced by injection of noradrenaline into the 3rdV of adult male Holtzman rats (250-300 g) implanted with a chronic stainless steel cannula into the 3rdV. The injection volume was always 1 mu l and was injected over a period of 30-60 sec. Control animals were injected with 0.15 M NaCl. After the injection of isotonic saline (control, 0.15 M NaCl) into the 3rdV, water ingestion was 0.3 +/- 0.1 ml/h. Ramipril (1 mu g/mu l) injected into the 3rdV prior to isotonic saline produced no changes in water ingestion (0.4 +/- 0.2 ml/h). The injection of noradrenaline (40 nmol/mu l) after isotonic saline induced an increase in water intake (3.0 +/- 1.1 ml/h). The prior injection of ramipril decreased this ingestion to 1.8 +/- 0.3 ml/h. These data show that the inhibition of converting enzyme in the brain reduces the water intake induced by catecholaminergic stimulation. We conclude that the brain is able to transform the prodrug ramipril into the active drug ramiprilat.

Enhancement of natural killer cell function by titanocenes in mice bearing Ehrlich ascites tumour

In the present work, we studied the effects of two titanocenes, biscyclopentadienyidichlorotitanium IV, (DDCT) and its derivative, biscyclopentadienylditiocianatetitanium IV (BCDT), on the activity of natural killer (NK) cells in Ehrlich ascites tumour (EAT)-bearing BALB/c mice. In order to investigate a more direct effect of these compounds on NK cell function, we performed experiments with severe combined immunodeficiency (SCID) mice, which exhibit a normal NK cell response in the absence of T and B cells. The treatment consisted of intraperitoneal (i.p.) administration of 15 mg/kg/day of DDCT for 2 days or 10 mg/kg/day of BCDT for 3 days. In addition, to verify whether the effects produced by the titanocenes were compound specific or related to a direct antitumour effect, we also investigated the effects of a 3-day treatment with 100 mg/kg of cyclophosphamide cyclophosphamide on NK cell activity. Our results demonstrated that, in BALB/c and SCID mice, NK cell function declined to subnormal levels after inoculation of the tumour. In these animals, although treatment with DDCT and BCDT significantly enhanced NK cell function, only DDCT restored NK cell activity to normal values in all stages studied. Conversely, treatment with cyclophosphamide reduced NK cell function in nontumour bearing SCID mice and was also unable to restore the decreased NK activity of tumour-bearing SCID mice, thus demonstrating that the enhancement of NK cell function by titanocenes is compound specific. The same effect of cyclophosphamide was observed with BALB/c mice. In the present study, the up-modulatory effects of these two compounds on NK cell function reveal a new aspect of the mechanism of antitumoural action of titanocenes. (C) 2003 Elsevier B.V. All rights reserved.

Hypothalamic melanin-concentrating hormone is induced by cold exposure and participates in the control of energy expenditure in rats

Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.

Placental glutathione S-transferase correlates with cellular proliferation during rat tonguie carcinogenesis induced by 4-nitroquinoline 1-oxide

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure (p < 0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion. (c) 2007 Published by Elsevier GmbH.