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By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the lesion and lethal properties of the native components. Allergen extracts can be turned anallergic. Physiological proteins with strong biological activity can also be rendered innocuous. Tyrosine and histidine are the reactive groups that incorporate the hapten. Within the same batch of protein, there is a defined ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easily prepared, can be accurately standardized and have unlimited shelf lives. The derivatives are obtained in a short time. The cost of the entire procedure is very low. The method was applied to tityustoxin and whole venom of the scorpion Tityus serrulatus; crotoxin and whole venom of Crotalus durissus terrificus; to five bothropic venoms; to allergenic extracts of Schistosoma mansoni; to cholera and tetanus toxin; and to insulin, kallikrein and tonin. The derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected into animals. No local or systemic side effects were observed, even after prolonged use. The injections were also apparently painless. By extensive haptenization self proteins can be rendered non-self, able to generate antibodies against both the derivative and the native unmodified protein, and iodination was very convenient for this purpose. A new schedule for immunization, only feasible with completely toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of antibodies against the native antigen, in the generated sera is unmatched by other procedures.

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Theoretical considerations were advanced on the reaction rate of biological systems in a rocket accelerated at fractional levels of the velocity of light. The values of mass increase in reacting molecules and length contraction of space under these relativistic velocities attained by the hypothetical rocket were inserted in equations of the absolute reaction rate theory. The equations employed were for the frequency of collisions, and for the internal kinetic energy of molecular reactions. Results of both sets of equations indicated that reduction of reaction rates were correlated to the mass increase. This would imply a general slowing of all chemical, biochemical and biological processes taking place. A human would suffer a related decrease in metabolic rate. Contrary to what is generally accepted, the biological aging of the space traveler under velocities bearable by humans, namely under 0.50c, would follow a pace very similar to that of an observer remaining in the resting frame of reference. With increased increments of the velocity, the space traveler would display a more intense lowering of the metabolic rate, with signs and symptoms comparable to body core hypothermia. Metabolic rates at insufficient levels to maintain the vital functions would be attained at 0.70c and higher, leading swiftly to coma and death. The presence of an endocrine dysfunction such as hypothyroidism or obesity in the space traveler would aggravate the signs and symptoms. Space travel at efficient velocities would be unbearable for a warm-blooded animal.

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The venom from the snake Crotalus durissus terrificus was detoxified by stepwise incorporation of stable cationic iodine. The venoid, in a dosage equivalent to 100 LD50 of the lethal venom, was injected in mice without lethal exits. The native venom (NAT), or its toxoided (TXD) derivative were incubated in presence or absence of mitogens, with human mononuclear (MN), B and T cells, with a pulse of [3H]Thymidine. No synergistic or antagonistic effects were observed in the combined activity of the mitogens and NAT or TXD. In the direct action of NAT the incorporation of radioactivity into MN and T cells diminished with venom increase in concentration indicating that the cytotoxicity of the native venom was correlated with the amount added. With B cells, the native venom exercised an initial mitogenic activity, declining in the higher concentration. On the other hand, the TXD showed a consistent effect, increasing the thymidine uptake in a manner related to concentration. This stimulation by TXD was observed with all groups of cells. The results indicate that, by abolishing direct cytotoxic activity with toxoiding of this venom, a derivative that enhances mitogenesis in these white cells can be obtained.

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Fraction T2 from Tityus serrulatus venom produced a marked lengthening of action potentials recorded with the single sucrose-gap technique, a characteristic effect of alpha scorpion toxins. This effect was not reversed by thorough washing of the nerve. On the other hand, T2 fraction deactivated by complete iodination did not cause any alteration of the compound action potential, even if applied in concentration as high as 4000 times the half saturation dose of the unmodified T2 fraction. This high dose of deactivated T2 did not hinder the onset of the full effect of a single dose of T2 fraction applied subsequently. Polyclonal antibodies against native or against iodinated venom reverted the action of T2 fraction, restoring normal electrical response. We conclude that both types of antibodies may remove the effect of alpha toxins on sodium channel.

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Purified samples of tetanus toxin were gradually iodinated by stepwise addition of iodine, up to saturation. The residual capacity of each sample to provoke tetanus was tested by injecting sc 25 Lf (Limit of flocculation) in mice. Toxicity diminished in relation to iodine incorporated, and with iodine-saturated samples, doses of up to 100 Lf in mice, or 500 Lf in guinea-pigs, proved innocuous. Mice immunized with two doses of this toxoid adsorbed on Al(OH)3, and challenged with standard lethal toxin, gained protection against 10 MLD (Minimum Lethal Doses). Guinea-pigs were immunized by a single sc dose of 27.5 Lf with fluid toxoid, and all resisted challenge against 10 MLD applied 30 days later. The mice sera gave strong immunoprecipitation lines against the native toxin. The findings indicated that by controlled iodination of tetanus toxin an effective and inexpensive toxoid can be prepared.

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The acetylcholine (ACh) liberating effect on rat brain slices of tityustoxin, an alpha toxin from the scorpion Tityus serrulatus venom, was measured in the absence and presence of dithiothreitol (DTT). The rate of net ACh liberation by toxin concentrations of 2 nmol in 5 ml of organ bath, was 7.5 +/- 0.09 nmol g-1 min-1. If DTT at a final concentration of 1 mM was added after a 10-min incubation period with toxin alone, inhibition of tityustoxin activity was 94%. With DTT 0.1 or 0.01 mmolar inhibition was 74% and 57%, respectively. The intense secretagogue effect, both in salivary and pancreatic glands of adult rats, induced by sublethal doses of tityustoxin was not affected by i.v. injection 10 min later of DTT 1 mumol g-1 of rat weight. When tityustoxin was injected i.p. at a dose 3 times the LD50 in mice, death ensued in 40 to 60 min. If toxin inoculation in mice was followed 10 min later by DTT 1 mumol g-1 of mouse weight, injected i.p. or i.v., deaths were delayed to 90 to 110 min, but no survival was observed. At necropsy, none of the mice treated with DTT showed any signs of pulmonary edema.

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A tetraiodinated derivative of bovine insulin, prepared at pH 1 with stable iodine, was unable to cause signs of hypoglycemia in doses up to 2.4 micrograms/g in fasting mice, when native insulin caused 100% mortality. In neutral and acidic solutions, in absence of chaotropic agents, it behaved as the monomer, and could be separated from less iodinated, active species, that appeared as dimers, by conventional gel filtration. To generate antibodies in guinea-pigs, the tetraiodinated insulin was injected in doses three times higher than native insulin, without any harm to recipient animals. The induced antiserum was compared with antiserum generated by conventional methods in radioimmunoassay (RIA) of native insulin, and parallel curves were obtained.

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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.

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Iodinated kallikrein appeared inactive on synthetic substrates, was unable to liberate kinins from rat plasma, did not cause contraction of the rat uterus and did not potentiate bradykinin activity. After several doses of iodinated kallikrein were applied to the muscle, the contraction caused by kallikrein was blocked, but the response of the preparation to bradykinin was unaltered. If a double dose of kallikrein was applied, the contraction appeared again, and the response to bradykinin was also potentiated. These results support a kinin-mediated oxytocic action of kallikrein, and suggest a new interpretation for the mechanism of kallikrein desensitization in this preparation.

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Using monovalent organic mercurials to modify the response to the oxytocic action of bradykinin (BK) on the rat uterus, a charge effect, related to the reagent applied, was noted. With neutral, and positively charged mercurials, a bimodal pattern of activity, causing stimulation of BK contraction at lower, and inhibition at higher concentrations was observed. With negatively charged mercurials transient stimulation occurred, inhibition only appearing after very large doses and long periods of incubation. Anionic charges of the bradykinin receptor affect the ligand properties of mercurials, repelling negative charges. By themselves, mercurials did not cause any response.

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An inactive form of kallikrein prepared by iodination with cold iodine, did not show any enzymatic or oxytocic action. However, a competitive pattern between this inactive and active kallikrein was observed in rat uterus preparation: When the inactive form was applied several times in the muscle, a single dose of active kallikrein was unable to cause contraction, but a double dose elicited a response. The rhythmic movement caused by a singular dose of active kallikrein, had its time curtailed by adding the inactive kallikrein to the bath. The inactive kallikrein did not interfere with bradykinin activity.

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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.

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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

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Samples of Schistosoma mansoni soluble adult worm proteins (SWAP) were iodinated with 4-15 mumol I/mg protein using iodine monochloride. The capacity to elicit immediate hypersensitivity reactions of the iodinated derivatives was compared to that of the native SWAP preparations. The degranulation of mast cells from infected mice decreased with increasing iodine incorporation and was absent in fully iodinated samples containing 15 mumol I/mg protein. The response of guinea pigs and humans to the intradermal test with iodinated SWAP also decreased in proportion to iodine incorporation, and no responses were obtained with fully iodinated samples. No false-positive tests were observed. Antibodies to the fully iodinated extracts generated in C57BL/10 mice reacted by ELISA with the unmodified proteins and by immunoprecipitation on agar gel. The immunoprecipitation pattern suggested that some epitopes were altered by iodination.

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Samples of Schistosoma mansoni soluble adult worm proteins (SWAP) were iodinated with 4-15 µmolI/mg protein using iodine monochloride. The capacity to elicit immediate hypersensitivity reactions of the iodinated derivatives was compared to that of the native SWAP preparations. The degranulation of mast clls from infected mice decreased with increasing iodine incorporation and was absent in fully iodinated samples containing 15 µmol I/mg protein. The response of guinea pigs and humans to the intradermal test with iodinated SWAP also decrease in proportion to iodine incorporation, and no responses were obtained with fully iodinated samples. No false-positive tests were observed. Antibodies to the folly iodinated extracts generated in C57BL/10 mice reacted by ELISA with the unmodified proteins and by immunoprecipitation on agar gel. The immunoprecicpitation pattern suggested that some epitopes were altered by iodination

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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.

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1. Whole soluble venom from the snake Crotalus durissus terrificus was detoxified by controlled iodination. Doses equivalent to 100 LD50 of the native venom were administered to mice, without signs of intoxication. 2. The non-toxic iodinated derivatives were able to stimulate antibodies in rabbits and horses within a short period (6 months) of immunization. Horse antisera attained titers of 0.5 to 0.9 mg/ml for protection against native venom. 3. Horse antisera obtained in horses from native and iodinated venom were run against both native and iodinated venoms, as antigens, in gel immunodiffusion. The precipitation lines showed total identity of the two types of sera.

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1. Whole soluble venom from the snake Crotalus durissus terrificus was detoxified by controlled iodination. Doses equivalent to 100 LD50 of the native venom were administerd to mice, without signs of intoxication. 2. The non-toxic iodinated derivative were able to stimulate antibodies in rabbits and horses within a short period (6 months) of inmunization. Horse antisera attained titers of 0.5 to 0.9 mg/ml for protection aginst native venom. 3. Horse antisera obrained in horses from native and iodinated venom were run against both native and iodinated venoms, as antigens, in gel immunodiffusion. The precipitation lines showed total identity of the types of sera

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The iodination of the T2 fraction abolished its lethal capacity, and doses up to 30 times the LD50 were injected i.p. in mice without noticeable toxic effects. The modified fraction retained its immunological properties. Antibodies generated against the iodinated T2 fraction were also reactive toward the native T2 fraction, T1 fraction and the whole soluble venom.

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