Acute and chronic toxicological studies of Dimorphandra mollis in experimental animals.

Dimorphandra mollis Benth. (Caesalpiniaceae), known as "faveira" or "fava d'anta" is a common plant in central Brazilian cerrado that is used mainly as a vasoprotector. Its main marker is rutin. The present study aimed to evaluate the security of Dimorphandra mollis dry extract in rodents. The extract presented a rutin content of 76+/-3%. Acute and chronic (180 days) toxicity was evaluated after per os administration. In acute toxicity, 3500 and 5000 mg/kg doses presented reversible effects. In chronic toxicity, 1000 and 2000 mg/kg doses did not provoke significant changes in body weight of the animals and in water and food consumption. Behavioral reversible changes and in blood count parameters (hemoglobin, hematocrit and red cells decrease and platelets increase in male in rats) were observed only in 2000 mg/kg dose. In biochemical evaluation, the results varied a lot considering doses and sex, without a linear profile. Some parameters showed a significant difference but without a clinical correlation. In histopathological examination, lung hemorrhage was observed in 2000 mg/kg dose. In conclusion, the study suggest that the extract is safe in a 1000 mg/kg dose, whereas for 2000 mg/kg dose further studies are needed. In long-term use, caution is required.

Selective inhibitors of cyclo-oxygenase-2 (COX-2) induce hypoalgesia in a rat paw model of inflammation.

1. It is well-established that inhibitors of cyclo-oxygenase (COX) and hence of prostaglandin (PG) biosynthesis reverse inflammatory hyperalgesia and oedema in both human and animal models of inflammatory pain. 2. Paw oedema and hyperalgesia in rats were induced by injecting carrageenan (250 micro g paw(-1)) into a hindpaw. Both inflammatory responses were followed for 24 h after the injection, measuring hyperalgesia by decreased pain threshold in the paws and oedema by plethysmography. 3. Three selective inhibitors of cyclo-oxygenase-2 (COX-2), celecoxib, rofecoxib and SC 236, given systemically in a range of doses, before the inflammatory stimulus, abolished carrageenan-induced hyperalgesia with little reduction of oedema. These inhibitors also induced hypoalgesia, increasing nociceptive thresholds in the inflamed paw above normal, non-inflamed levels. This hypoalgesia was lost at the higher doses of the selective inhibitors, although hyperalgesia was still prevented. 4. In paws injected with saline only, celecoxib, given at the dose inducing the maximum hypoalgesia after carrageenan, did not alter the nociceptive thresholds. 5. Two non-selective inhibitors of COX-2, indomethacin and piroxicam, abolished hyperalgesia and reduced oedema but did not induce hypoalgesia. 6. Celecoxib given locally into the paw also abolished inflammatory hyperalgesia and induced hypoalgesia without reducing oedema. 7. We conclude that hypoalgesia is expressed only over a critical range of COX-2 inhibition and that concomitant inhibition of COX-1 prevents expression of hypoalgesia, although hyperalgesia is still prevented. 8 Our results suggest a novel anti-nociceptive pathway mediating hypoalgesia, involving COX-2 selectively and having a clear peripheral component. This peripheral component can be further explored for therapeutic purposes.

Aminoguanidine prevents impaired healing and deficient angiogenesis in diabetic rats.

The diabetic organism is unable to produce normal amount of granulation tissue which results in delayed wound healing, a significant clinical problem. In the present study, the effect of oral administration of aminoguanidine (AG), in the diabetes-induced inhibition of angiogenesis and granulation tissue formation was tested. Subcutaneous implantation of sponge discs in nondiabetic rats induced a wound repair response as determined by the amount of hemoglobin (vascular index) and granulation tissue formation (morphometric analysis) of the implants. In the streptozotocin-induced diabetic rats the predominant response indicative of healing was inhibitory. Aminoguanidine was effective in preventing in 50% the diabetes-induced inhibition of fibrovascular tissue growth in the implants, as indicated by the values of hemoglobin content and vascular growth areas of the implants. These results indicate that AG holds potential therapeutic value in the management of healing impairment of the diabetic condition.

Pharmacological evidence supporting a role for IL-1, IL-2 and serotonin in the inflammation induced by Schistosoma mansoni soluble egg antigen (SEA) in rat paws.

THIS study intended to characterize pharmacologically the mediator(s) released in the inflammation induced by Soluble Egg Antigen (SEA), the main antigen released from eggs of Schistosoma mansoni, in rat hindpaws. A single intraplantar injection of 0.1-100 microg SEA at day zero induced a dose-dependent increase in the volume of rat hindpaws characterizing an oedema of quick onset (within 15 min) and 4h-duration, which was confirmed by histopathological analysis of the paws. A second injection of SEA in the same paw (1-10 microg) 28 days later induced an increased dose-dependent oedematogenic response. The early oedematogenic response following SEA sensitization was derived from serotonin release and interleukin-1 (IL-1), since treatment with either pizotifen or an antibody against IL-1, reduced the response by 60% and 48%, respectively. The increased oedematogenic response derived from SEA-challenge (10 microg) of rat paws derived from a local rather than systemic reaction, since it was not observed if the sensitization was in the contralateral paw or the peritoneal cavity of the animals. Chronic treatment with inhibitors of IL-2 synthesis/release such as cyclosporin or dexamethasone during the sensitization phase reduced the oedematogenic response due to SEA challenge by 51% and 55%, respectively. These data suggested that SEA-challenge was immune-derived and dependent of IL-2 release. It is discussed the association between cytokine release and the resistance of rats to S. mansoni infection.

Lung oedema induced by Tityus serrulatus scorpion venom in the rat.

The effects of drugs were investigated on the induction of acute lung oedema by scorpion Tityus serrulatus venom in male Wistar rats (200-230 g) anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). Intravenous (i.v.) injection of scorpion venom (0.5 mg/kg) into 12 rats induced arterial hypertension and severe lung oedema, whereas i.v. injection of scorpion venom into 16 rats previously injected with commercial heparin induced arterial hypertension, but only a slight lung oedema. It is suggested that the inhibitory effect of commercial heparin on the genesis of lung oedema may be due to a decrease in vascular permeability in the lungs. Previous i.v. injection of aprotinin did not prevent the arterial hypertension and the lung oedema induced by scorpion venom. Previous injections of platelet-activating factor antagonists (BN-52021 and WEB-2170) or of an inhibitor of lipo- and cyclooxygenase (Nordihydroguaiaretic acid) did not prevent the arterial hypertension induced by scorpion venom, but decreased the magnitude of the lung oedema elicited by the venom. Previous injections of inhibitors of 5-lipoxygenase (MK-886) or cyclooxygenase (aspirin or indomethacin) significantly decreased the magnitude of the lung oedema induced by scorpion venom. It is concluded that the release of vascular permeability factors, such as platelet-activating factors, leukotrienes, and prostaglandins may play a role in the induction of acute lung oedema by scorpion venom in rats.

Pharmacological characterization of Sephadex-induced oedema in rat paws: predominant role of serotonin and platelet-activating factor.

An intravenous injection of Sephadex beads has been used to induce lung inflammation and bronchial hyperreactivity in small animals. In the present study, we injected Sephadex beads (0.3-5.5 mg/paw) into rat paws and followed the resulting inflammation plethysmometrically. Our results show that Sephadex beads induced a significant and dose-dependent increase in the hindpaw volume at 5 min; it was maximal at 30-60 min and declined at 4 h. However, the paw volume remained significantly increased for up to 21 days. The initial 4-hour-oedema was confirmed by histopathology of the paw tissues, but the persistent increase in paw volume was related to a chronic inflammatory (granulomatous) response. The Sephadex-induced oedema was predominantly due to serotonin (5-HT) release since specific antagonists such as methysergide (1 mg/kg) and pizotifen (0.1-2 mg/kg) administered both systemically and locally were able to inhibit the oedema (10-100 microgram/paw) as could pretreatment with compound 48/80. In addition, platelet-activating factor (PAF) was also shown to be involved, since systemic pretreatment using the specific PAF antagonist BN 52021 (1 mg/kg) was able to inhibit the increase in paw volume induced by Sephadex. Effective doses of indomethacin (2 mg/kg), L-NAME (1 mg/kg), pyrilamine (1-2 mg/kg), ondansetron (1 mg/kg) and HOE 140 (1 mg/kg) did not affect the Sephadex-induced oedema, thus ruling out the participation of prostaglandins, nitric oxide, histamine, 5-HT3 receptors and bradykinin in its development. Since the late increases in paw volume induced by Sephadex were reduced by pretreatment of the animals with the immunosuppressive drugs rapamycin and dexamethasone but not cyclosporin, our results also suggested that distinct immunological pathways may be involved in the modulation of the chronic phase of inflammation induced by Sephadex beads in rat paws.

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice

Non-viable cells and biochemical fractions from Paracoccidioides brasiliens were obtained for experimental inoculation in mice and posterior histopatological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arised from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitonially inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflamatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice.

Non-viable cells and biochemical fractions from Paracoccidioides brasiliensis were obtained for experimental inoculation in mice and posterior histopathological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arose from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitoneally inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflammatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed.

A nontoxic derivative of Bothrops jararaca venom suitable to generate antibodies against the native venom.

Bothrops jararaca venom was toxoided by stepwise iodination with cold iodine, and doses up to 30 LD50 were non-lethal by i.p. route (BICALHO et al., 1990). Groups of mice injected chronically with the native, or the iodinated venom, have been subjected to histological examination. In the native group, in the spleen, around the white pulp, an acellular, amorphous eosinophilic substance, metachromatic to Methyl Violet, PAS positive, and dichroic to Congo Red under polarized light, was present. Strong congestion in the liver, kidneys and lungs was found. The salivary glands were replenished with an amorphous substance in the serosal acini. The groups injected with the iodinated venom only show discrete alterations, more akin to the control group. The anavenin was immunogenic. Antibody generation in mice and rabbits was detected by ELISA. In mice, active protection against challenge with native venom was presented. The iodinated venom generated a rabbit antiserum with strong lines in gel immunoprecipitation against the lethal venom. A minimum neutralization titer of 2.3 mg ml-1 against the native venom was attained in the first cycle (28 days) of immunization. After 3 cycles (100 days), the protection rose to 5.1 mg ml-1.

A toxoid prepared from cholera toxin by iodination.

A cholera toxoid was prepared by iodinating purified cholera toxin having an activity of 25 Limit of blueing (Lb) doses/1 microgram with 0.8 mumol of iodine monochloride per mg toxin, and the residual lesion capacity was tested in mice. The blueing dose (BD) test was strongly positive for the native toxin, and completely abolished in the iodinated toxoid when tested at up to 25 times on Lb dose. The dermal microscopic lesions with intradermal doses of 1 microgram virulent toxin presented intense leucocyte infiltration, proteinaceous edema and active hyperemia, whereas none of these effects was observed with the same amount of toxoid. To determine antigenicity, two groups of mice received toxin or toxoid, 8.5 micrograms adsorbed to aluminum hydroxide, followed by a booster of 17 micrograms in saline 21 days later. Measurement of antibodies by ELISA at day 28 indicated that the toxoid was 2.5 times more antigenic than the toxin. These data show that iodination converts cholera toxin to an effective toxoid.

A toxoid prepared from cholera toxin by iodination

A cholera toxoid was prepared by iodinating purified cholera toxin having an activity of 25 Limit of blueing (Lb) doses/l ug with 0.8 umol of iodine monochloride per mg toxin, and the residual lesion capacity was tested in mice. The Blueing Dose (BD) test was strongly positive for the native toxin, and co0mpletely abolished in the iodinated toxoid when tested at up to 25 times one Lb dose. The dermal microscopic lesions with intradermal doses of 1 ug virulent toxin presented intense leucocyte infiltration, proteinaceous edema and active hypertemia, whereas none of these effects was observed with the same amount of toxoid. To determine antigenicity, two groups of micereceived toxin or toxoid, 8.5 ug adsorbed to aluminum hydroxide, followed by a booster of 17 ug in saline 21 days later. Measurement of antibodies by ELISA at day 28 indicated that the toxoid was 2.5 times more antigenic than the toxin. These data show iodination converts cholera toxin to an effective toxoid

The effect of stepwise iodination on biological properties of Bothrops jararaca venom.

By titrating 5 mg of native venom with aliquots of a 2 x 10(-2) M iodine monochloride solution, neutralization of lethality by the incorporation of iodine was found with 200 +/- 5 microliters of solution, and above, up to 310 +/- 10 microliters, when saturation with iodine was attained. Doses up to 1500 micrograms (equivalent to 32 LD50 of native venom), where injected i.p. in mice without lethal effects. Proteolytic, phospholipase A2 and esterolytic activities were greatly reduced, but a low activity persisted even in fully iodinated samples. Direct hemolysis was markedly inhibited, and incapacity to coagulate fibrinogen and horse plasma was also observed in the iodinated samples. Hemorrhage and necrosis in rat skin, caused by 20 micrograms of iodinated venom were not elicited by doses up to 120 micrograms of iodinated anavenom. In mice, the myonecrosis that resulted from direct i.m. injection of native venom, and the massive hemorrhage caused by 5 LD50 doses injected i.p. were abolished by venom iodination. Blood congestion in liver, spleen, kidneys, and lungs, almost disappeared with iodination to the level of neutralization, and was barely seen with venom samples iodinated to saturation. The clinical signs of impaired physical activity, appearing in mice injected with 700 to 1500 micrograms of the iodinated anavenom were intensified by captopril and attenuated by epinephrine.

Effect of granulocyte inhibitory factor on granuloma formation in mice infected with Schistosoma mansoni.

Mononuclear cells from patients infected with Schistosoma mansoni were able to produce a soluble material that inhibited the granulocyte cytotoxicity against schistosomula in a complement-dependent killing assay. This granulocyte inhibitory factor (GIF) appears to exist preformed in the mononuclear cells of patients, but it can also be released in the supernatant after antigenic stimulation (lymphokine-like). Only T lymphocytes were able to mediate the inhibition of granulocyte cytotoxicity against schistosomula in vitro. The treatment of S. mansoni-infected mice with GIF induced a significant decrease in the liver granuloma size.

Efeito da timectomia neonatal na esquistossomose mansoni experimental. / Effect of neonatal thymectomy in experimental schistosomiasis mansoni

Camundongos albinos recem-nascidos foram submetidos a timectomia e infectados 30 dias apos com 60 cercarias de Shistosoma mansoni. Foi incluido no experimento um grupo controle constituido de animais timectomizados, nao infectados, nao timectomizados infectados e nao timectomizados nao infectados. Foi realizado estudo histopatologico minucioso dos animais antes do inicio da oviposicao e sete semanas apos a infeccao. Os animais timectomizados e infectados mostraram quadro caracteristico devido a deplecao dos linfocitos T, com marcada diminuicao do desenvolvimento do granuloma e escasso exsudato inflamatorio em torno dos ovos. Lesoes graves foram observadas no figado, em animais sacrificados sete semanas apos a infeccao e sem qualquer reacao inflamatoria. Foram muito escassos os fenomenos exsudativos intra e extralobulares, no figado. Os fenomenos necroticos degenerativos dos hepatocitos foram considerados como sendo devidos a infeccao esquistossomotica, em animais com deplecao de linfocitos T