As “MEMÓRIAS DO INSTITUTO BUTANTAN’’ têm por finalidade

a apresentação de trabalhos originais que contribuam para o progresso nos

campos das Ciências Biológicas, Médicas e Químicas, elaborados por especia¬

listas nacionais e estrangeiros.

São publicadas sob a orientação da Comissão Editorial, sendo que os

conceitos emitidos são de inteira responsabilidade dos autores.

The “MEMÓRIAS DO INSTITUTO BUTANTAN” are the vehicle of

communication for original papers written by national and foreign specialists

who contribute to the progress of Biological, Medicai and Chemical Sciences.

They are published under the direction of the Editorial Board which as¬

sumes no responsability for statements and opinions advanced by contribu¬

to rs.

Diretor do Instituto Butantan

Dr. Willy Beçak

Comissão Editorial

Henrique Moisés Canter — Presidente

Adolpho Brunner Júnior — Membros

Raymond Zelnik

Saul Schenberg

Sylvia Lucas

Denise Maria Mariotti —- Bibliotecária

Periodicidade: irregular

Permuta/ Exchange: são feitas entre entidades governamentais, com publica¬

ções congêneres, mediante consulta prévia. Exchanges with similar publica-

tions can be settled with academic and governamental institutions through

prior mutual agreement.

Endereço/Address: Instituto Butantan — Biblioteca. Av. Vital Brasil, 1.500.

CP 65 — 05504 — São Paulo, SP — Brasil.

Telefone/Telephone: (011)211-8211 — R. 129 — Telex: 1121325 BUTA-BR.

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10 11 12 13 14 15 16

Governo do Estado de São Paulo

Secretaria de Estado da Saúde

Coordenação dos Institutos de Pesquisa

Instituto Butantan — São Paulo — SP — Brasil

MEMÓRIAS

INSTITUTO BUTANTAN

Volume 49, número 1,1987

Sao Paulo, SP— Brasil

1987

MEMÓRIAS do INSTITUTO BUTANTAN. (Secretaria de Estado da Saúde)

São Paulo, SP — Brasil, 1918 —

1918 - 1983/84, 1 - 47/48

Publicação interrompida de 1985 a 1986.

1987, 49(1

ISSN 0073-9901

MIBUAH CDD 614.07205

Solicita-se permuta / Exchange desired

í, | SciELO

Mem. Inst. Butantan

49 ( 1 ), 1987

SUMÁRIO / SUMMARY

EDITORIAL

Mudança na publicação de Memórias do Instituto Butantan e

Coletânea de Trabalhos do Instituto Butantan.

A change in publication of Memórias do Instituto Butantan

and Coletânea de Trabalhos do Instituto Butantan.

History of the primordia of snake bite accident serotherapy.

História dos primórdios da soroterapia antiofídica.

Oswaldo VITAL BRAZIL. 7-20

Avaliação "in vivo" da vacina bivalente contra a gripe, produzida no

Instituto Butantan.

Evaluation "in vivo" of influenza vaccine, produced at Instituto Bu¬

tantan.

Dalva A. Portari MANCINI; Cirlene A. VILELA; Mariangela M.

MOREIRA. 21-24

Spider venoms acting on the sodium channel.

Peçonhas de aranhas que atuam no canal do sódio.

Oswaldo VITAL BRAZIL. .

25-33

7 SCÍELO, 1:l 12 13 14 15 16 17

Mem. Inst. Butantan

49(11, 1987

EDITORIAL

MUDANÇA NA PUBLICAÇÃO DE MEMÓRIAS DO INSTITUTO BU¬

TANTAN E COLETÂNEA DE TRABALHOS DO INSTITUTO BUTAN¬

TAN.

A CHANGE IN PUBLICATION OF MEMÓRIAS DO INSTITUTO BU¬

TANTAN AND COLETÂNEA DE TRABALHOS DO INSTITUTO BU¬

TANTAN.

A partir do volume 49, 1987, as “MEMÓRIAS DO INSTITUTO BU¬

TANTAN" serão editadas em fascículos, em intervalos irregulares.

A "COLETÂNEA DE TRABALHOS DO INSTITUTO BUTANTAN"

não mais será editada como volume independente, passando a ser incluída,

sob a forma de resumos, no último fascículo das "MEMÓRIAS DO INSTI¬

TUTO BUTANTAN", com o título de "COLETÂNEA DE RESUMOS DE

TRABALHOS PUBLICADOS PELOS PESQUISADORES DO INSTITUTO

BUTANTAN".

As of volume 49, 1987, the "MEMÓRIAS DO INSTITUTO BUTAN¬

TAN" will furtheron be edited in fascicules at irregular intervals.

The "COLETÂNEA DE TRABALHOS DO INSTITUTO BUTAN¬

TAN" will no more be edited as independent volumes, but will be included

in the last fascicule of the "MEMÓRIAS DO INSTITUTO BUTANTAN" as

abstracts under the title "COLETÂNEA DE RESUMOS DE TRABALHOS

PUBLICADOS PELOS PESQUISADORES DO INSTITUTO BUTANTAN".

Mem. Inst. Butantan

49 ( 1 ): 7 - 20 , 1987

HISTORY OF THE PRIMORDIAOF

SNAKE-BITE ACCIDENT SEROTHERAPY*

Oswaldo VITAL BRAZIL**

First I would like to thank the organizers of this session in honor of Vital

Brazil, the founder of this Institute, for the invitation to address you on his

life and scientific work. I believe this was motivated by the fact that not

only I am one of his sons but also a scientist belonging to his scientific

school and one of the continuators in this country of his researches on the

South American snake, scorpion and spider venoms. To present, even suc-

cinctly, all of Vital BraziFs accomplishments in an exposition which must

not be overly lengthy seemed to me impossible. I shall restrict myself, the-

refore, to present a historical summary on antivenomous serotherapy and

to make, therefore, an exposition of the research that marked so deeply the

destiny of this Institute, one of our country's most important and humani-

tarian scientific institution and certainly the most original.

The introduction of serotherapy in the treatment of snake-bite acci-

dents was mainly due to two scientists, Albert Calmette and Vital Brazil.

The former demonstrated in 1884 that the serum from animais immunized

with snake venoms was capable of neutralizing them, thereby making pos-

sible its preventive and curative application to counteract their effects in

the animal organism. Starting from this verification, Calmette immunized

horses with snake venoms and the antivenin or "serum antivenimeux", as

he called it, was distributed for use in the treatment of accidents caused by

snakes in various parts of the world, in particular in Indochina, India, Aus¬

trália and Europe. Vital Brazil was the first to demonstrate the specificity of

the antivenins, a fact which paradoxically was neither recognized nor ad-

mitted by Calmette. Following this discovery, Vital Brazil started in 1901 to

prepare mono and polyvalent antivenins — the anticrotalic, antibothropic

and antiophidic sera — for use in Brazil. Fie was, thus, the creator of the

antivenomous serotherapy on a really effective basis. His orientation —

preparation of mono and polyvalent antivenins for use in a determined re-

gion — is adopted world-wide today.

Translation of the address given during the session in honor of Vital BraziCs memory held in 28 April 1986 at the

Instituto Butantan.

Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, P.O. Box 6111, Campi¬

nas, São Paulo, Brazil.

VITAL BRAZIL, O. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49 ( 11 : 7 - 20 , 1987 ,

Albert Calmette

(1863 - 1933 )

Calmette 4 was born in 1863 in a very old and mountainous

country, the region of Auvergne, in the Massif Central of France. His ideal

as a boy: to be a naval officer and to take part in the civilizing mission of his

nation in distant regions. This ideal was unfulfilled for the benefit of Science

and humanity. A long illness contracted at the Lycée of Brest prevented

him of doing so. However, he joined Brest's naval School of Medicine in

1879. After being submitted to the required examinations in 1881, he beca-

me an auxiliary physician ("aide-médecin"). Then, he was sent to serve on

a war vessel in Annan and Tonkin (middle and north Vietnam). As a doctor

on board, he participated in heroic first aids during the bloody naval battles

between French ships and the Chinese fleet. Back to France, he submitted

himself successfully to all examinations, including defence of thesis, at the

Faculty of Medicine of Paris, to obtain his degree of medicai doctor. Fie

then went to serve his country in the torrid and humid Gabon on the west

coast of África. On returning to France, he married and managed to be de-

signated for a post where he could take with him his young wife: doctor on

the Islands of Saint Pierre and Miquelon, a land of fishermen, with an ex-

tremely rigorous climate, situated south of Newfoundland in the North

Sea. It was there that he began his self-taught study and practice of bacte-

riology, a new Science at that time, carrying out a research which enabled

him to be accepted by Roux in the course of Pasteur Institute and to beco-

me in the future one of the most illustrious pasteurians. After a training

program of three months at the Institute, he was chosen by Pasteur himself

to set-up and direct, in the then French colony of Indochina, a laboratory

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VITAL BR AZIL, 0. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

4901 : 7 - 20 , 1987.

for the preparation of vaccine which the incidence of 95% of smallpox in

the native population made urgent, and also of the antirabic vaccine. In Ja-

nuary of 1891, he leaved with his wife for the distant Cochinchina where, in

Saigon he founded the Pasteur lnstitute's first branch. A fortuitous occur-

rence in October 1891 awakened his attention to the problem of snake-bite

accidents and incited him to study snake venoms. A village in the vicinity of

Bac-Lieu, about two hundred kilometers from Saigon was invaded by nu-

merous cobras (Naja naja) fleeing from the inundation occurring there. The

snakes entered the houses; forty natives were bitten and four died in few

hours. An annamese, a combination of witch doctor and snake charmer,

managed to capture nineteen of them which were sent to the recently crea-

ted Bacteriological Institute of Saigon by the administrator of the region.

Fourteen arrived alive. Calmette sacrificed eleven in order to remove their

venomous glands and obtain their venom. With the venom from the

twenty two venomous glands he started in the laboratory at the Bacteriolo¬

gical Institute of Saigon his studies on snake venoms. A paper 14 was pu-

blished in the "Annales de 1'lnstitut Pasteur" in 1882 announcing favorable

results, never confirmed, with the use of gold chloride in the treatment of

animais injected with the venom. He stated in this paper that the repeated

injection of warmed or unwarmed venom confers to the experimental ani¬

mais a certain resistence to the venom, which was interpreted as sorpe sort

of mithridatism, not as a real State of immunity. Nonetheless, upon retur-

ning to Paris in 1893, he initiated at the laboratory of Roux in the Pasteur

Institute, a research on the immunity conferred on the laboratory animais

by the Naja and other snake venoms. The discovery by Behring and Kitasa-

to 3 in 1890 of the antitoxic immunity in relation to diphtheria and tetanus to-

xins certainly stimulated the investigation. The results of this research were

presented in February of 1894 to the "Societé de Biologie " ,5 and also pu-

blished in the same year in the Annales de 1'lnstitut Pasteur , 16 His conclu-

sions were correct, except one: the absence of specificity of the serum of

the immunized animais. "Le sérum des animaux immunisés est

antitoxique, préventif et therapeutique non seulement à 1'egard du venin

qui a servi à immuniser 1'animal, mais même à 1'egara des venins d'autres

origines" he affirmed in his communication to the "Societé de Biologie"

("the serum from the immunized animais is preventively and therapeuti-

cally antitoxic not only in reiation to the venom that was used to immunized

the animal but even in relation to venoms of other origins"). In the work

published in the "Annales de 1'lnstitut Pasteur": "le sérum d'un lapin immu-

nisé contre le venin de cobra ou de vipère agit indifferement sur tous les ve¬

nins que j'ai experimentés" ("The serum from a rabbit immunized against

the cobra or viper venom neutralizes as well all the venoms I have assa-

yed"). The immunization of two mules was attempted at the Pasteur Insti¬

tute where at the annex of Garches, thanks to a popular subscription, ade-

quated installations were constructed in this period for Roux to initiate the

production of the antidiphtheric serum, avidly required by the medicai class

of France and other countries for the treatment of croup. Production of the

"sérum antivenimeux" took place in the city of Lille, where on the recom-

mendation of Pasteur and Roux, Calmette set-up and directed for many

years, the Pasteur Institute of Lille, a laboratory designated to attend the

region of North France. Calmette never stated clearly which venoms he

VITAL BRAZIL, O. History of the primordia of snakebite accident serotherapy. Mem. Inst. Butantan,

49 ( 11 : 7 - 20 , 1987

used in the immunization of the horses.* It is certain, however, that the

main venom if not the only one (since he did not admit the specificity of the

antivenins) was that of Naja naja he received from Indochina. In subse-

quent years, several researches showed that Calmette's "sérum antiveni-

meux", although neutralizing the Naja naja venom, was incapable of doing

so in relation of the venoms of other snakes, even of those pertaining to the

Elapid family to which the Naja naja belongs. Martin, 24 professor of Physio-

logy at the University of Melbourne, later on director of Lister Institute in

London was the first or one of the first to disagree, in 1897, with Calmette's

statement that a hyperimmune serum in relation to cobra venom is able to

neutralize the venom of other snakes: 'This statement (affirmation that the

hyperimmune serum against Naja naja venom is equally efficient in neutrali¬

zing other snakes venoms) was surprising because Behring, from the exa-

mination of the relations of various toxins and antitoxins, had arrived at the

conclusion that the curative value of immunising serum was specific, i.e.,

distinct toxins require distinct antitoxins, and some actions of different

kinds of snake venom are quite as different as, say, the actions of the to¬

xins of tetanus and yellow fever". He formulated, however, the hypothe-

sis, also erroneous, that the specificity of antivenomous sera is due to the

presence in snakes venoms of two types of proteic constituints, one being

destroyed by heating to 75-85°C, of heavier molecular weight, predomi-

nant in the venom of the Viperidae, the other being resistent to the heating

at these temperatures, of lighter molecular weight, predominant in the ve¬

nom of the Elapidae. However, he did not immunize animais with these ve¬

nom constituints in order to verify the correctness of his hypothesis. It was

up to Vital Brazil to demonstrate the specificity of the antivenins by immu-

nizing animais separately with the venom of different snake species and ve-

rifying that the sera from these animais neutralized exclusively or with much

greater efficiency the venom which was used for the immunization. He was

also the first to prepare mono and polyvalent antivenins for use in a determi-

ned region.

Vital Brazil was born in 1865, two years, therefore,after Calmet-

te, in the little old town of Campanha on the highlands of the Mantiqueira,

south of Minas Gerais. His high-school studies were made in São Paulo

where his family settled residence when he was thirteen year old. Thereaf-

ter, he joined the Faculty of Medicine of Rio de Janeiro, one of the only

two that operated in Brazil in the last century. While a student of medicine,

he intended to study the venom of Brazilian snakes. He had to give up the

idea: he did not meet the least receptivity to it on the part of the professors

who had laboratories inthe Faculty in which the research might be made. In-

Brazil at that time and unfortunaíely for still many years, the Faculties were

considered to be only places of professionalizing education, never of re¬

search.

In 1881,-at the age of 26, Vital Brazil received the degree of medicai

doctor. He, then, returned to São Paulo and worked in the first year of

after-graduation as a doctor in the Police Force of the State of São Paulo.

"J'ai étudié á ce point de vue le Sérum de Lille tel qu'il est fourni par le commerce; mais on ne sait pas exactement

quels son les venins utilisés par Calmette â la préparation de son cheval á Serum", ("I have studied from this point.

of view the Lille's Serum as it is presented at the commerce; but it is not known exactly which venoms are used by

Calmette for immunizing the horses that furnish the Serum") (from a letter of Maurice Arthus to Vital Brazil dated

of February 11, 1911)

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VITAL BRAZIL, 0. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49(11:7-20, 1987.

Vital Brasil

(1865 - 1950)

Afterward he joined the Public Health Department of the States as a Sani-

tarian Inspector. At that time, public health in São Paulo had deteriorated

due to the abrupt increase in its population caused by the arrival of Euro-

pean immigrants, mainly from Italy, who were more susceptible to the en-

demic diseases, specially yellow fever, prevalent in the State. Besides they

brought some others unknown in the country such as cholera morbus, of

which a few outbreaks occured in São Paulo. Vital Brazil fulfilled with extre¬

me dedication and competence the various commissions for which he was

designated: to combat yellow fever in the hinterland of the State and cho¬

lera morbus in the valley of Parayba. For this gratification, his conduct in

the fulfillment of his duties were always exalted by his superiors in the Pu¬

blic Health Department. Already married, with a daughter and limited fi¬

nancial resources, he did not think it right to risk so much his life. Therefo-

re, he left the Public Health Department to practice medicine in the town of

Botucatu in the hinterland of São Paulo, then a pioneerland, being quite

successful. The idea of studying snake venoms once again occurred to

him, now suggested by the verification of the ineficience of the medicai re¬

sources to treat patients bitten by venomous snakes. He planned to investi-

gate whether the plants claimed by the people to be efficient in the treat-

ment of snake bite accidents would show any curative effect on enveno-

med laboratory animais. The results from these experiments were always

negative. Taking knowledge, then, of the studies of Calmette on snake ve-

nom immunity, he understood that the antivenomous serotherapy was the

right way to solve the problem of snake bite accident treatment. He deci-

ded thus, to return to São Paulo and to join the Bacteriological Institute

VITAL BR AZIL, O. History of the prlmordia of snake-bite accident serotherapy. Mem. In st. Butantan,

49(1 ):7-20, 1987

where he could count on resources to carry out the research on Brazilian

snake venoms and snake venom immunity.

The Bacteriological Institute, 18 founded in 1892, was the first laboratory

of bacteriology and parasitology in Brazil dedicated to public health pro-

blems. At the request of the Government of the State of São Paulo, Pas-

teur indicated the French bacteriologist Felix Le Dantec to set-up and direct

it. However, after only four months, Le Dantec resigned and returned to

Europe. He was substituted by Adolpho Lutz, already working at the Bac¬

teriological Institute, on the recommendation by Le Dantec himself. "He is

a Brazilian capable of directing the laboratory", he affirmed. Adolpho Lutz,

son of Swiss, was bom in Rio de Janeiro in 1855. He was raised, however,

in Europe where he received the degree of medicai doctor at the University

of Bern, Switzerland, in 1880. Lutz had a very solid medical-scientific back-

ground, mainly in the field of morphological Sciences, specially parasito¬

logy, entomology, mycology, zoology and pathology. He was a very com-

petent director of the Institute, studying and elucidating with his coworkers

at the laboratory the etiology and distribution in the State of various ende-

mic and epidemic diseases. Vital Brazil joined the Bacteriological Institute

in 1897 as an assistant. Lutz not only gave his consent to Vital Brazil to

carry out the research on snake venoms at the Institute but also his help in

solving some problems in its execution as, for instance, the most reliable

process of catching venomous snakes: "Tendo nós entrado para o Institu¬

to Bacteriológico (1897)", he wrote in 1901, 5 "onde tivemos permissão pa¬

ra continuar nossas pesquisas, vimos esta e outras dificuldades removidas

pelo nosso sábio mestre Dr. Adolpho Lutz, que imaginou diversos apare¬

lhos apreensores (de serpentes). Dentre eles o que melhores resultados

práticos deu, foi o que nós denominamos laço", ("having joined the Bacte¬

riological Institute (1897), where I had the permission to continue the re¬

search, this and other difficulties were done away with by Dr. Adolpho

Lutz who planned various apparatus of snake trap. Among them the one

we denominated as "laço" (lasso) gave the best results"). Vital Brazil ex-

tracted the venom from the most common venomous snakes in the State -

rattlesnake (Crotalus durissus terrificus), jararaca (Bothrops jararaca), uru¬

tu (B. alternatus ), jararacussu (B. jararacussu) — by the process still in use

at the Butantan Institute nowadays. The amount of venom obtained from

the different snake species was determined as well as their lethal doses in

pidgeons, rabbits, guinea-pigs and dogs. The signs and symptoms evoked

by the venoms on the experimental animais were faithfully described as

well as the macroscopic lesions found at the autopsy of the animais. For

the first time, the signs and symptoms as well as the lesions evoked by the

South American rattlesnake venom on one hand and by jararaca and urutu

venoms on the other were shown to be quite different. Immunization expe-

riments on dogs, goats, oxen and horses were carried out. It was found 6

that "o cão é animal muito resistente e facilmente imunizável" ("The dog is

an animal very resistant to snake venom and easily immunized") whereas

"o cabrito, o boi e o cavalo são muito mais sensíveis e só a custo de grande

trabalho e paciência consegue-se levar qualquer destes animais a um esta¬

do de imunização capaz de fornecer soro bastante ativo" (the goat, the ox

and the horse are much more sensitive to the venom and only at the expen-

se of much skill and patience does one manage to bring any one of these

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10 11 12 13 14 15 16

VITAL BRAZIL, O. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49(11:7-20, 1987,

animais to a State of high immunity"). Vital Brazil also found at this time

(1898) that the antevenins are specific: "Tendo imunizado um certo núme¬

ro de cães contra o veneno de cascavel e outros contra o de jararaca", he

wrote in 1901, 6 "conseguimos soros bastante ativos, tendo verificado que

o soro do animal imunizado contra o veneno de jararaca nenhuma ação ti¬

nha em relação ao da cascavel, bem como o soro muito ativo contra o ve¬

neno crotálico mostrava-se muito fraco em relação ao veneno da jararaca"

("Having immunized some dogs against rattlesnake venom and others

against jararaca venom, I obtained quite active sera, having found that the

serum from animais immunized against jararaca venom did not neutralize

that of rattlesnake, just as the very potent serum against the rattlesnake ve¬

nom was quite weak in neutralizing the jararaca venom"). In 1899 he had to

interrupt temporarily the research. "A mortandade de ratos em Santos e o

aparecimento de casos mórbidos que, por sua sintomatologia, tornaram-se

suspeitos de peste bubônica inspiraram a Diretoria Geral do Serviço Sanitá¬

rio a acertada providência de destacar para Santos um dos ajudantes do

Instituto Bacteriológico com o instrumental necessário para, na primeira

oportunidade, colher material de estudo e proceder a pesquisas bacterioló¬

gicas", he recorded in a report of December, 1889 on the outbreak of bu-

bonic plague in Santos. 7 ("Rat mortality in Santos as well as the occurence

of cases of an illness which by its symptomatology was suspected to be bu-

bonic plague, suggested the Administration of the Public Health Departa-

ment to send one of the Bacteriological lnstitute's assistants to Santos in

order to collect, in the first opportunity, material for study and to proceed

with bacteriological investigations"). Vital Brazil was indicated by Lutz for

this mission. "No dia 9 de outubro partimos para Santos", he reported "le¬

vando um microscópio, meios de cultura, pipetas, tubos esterilizados, fer¬

ros para autopsia etc. Instalamos nosso gabinete de observação em um

dos quartos do Hospital de Isolamento". ("On October 9, I left for Santos

taking a microscope, culture media, pipettes, sterilized tubes, autopsy ins-

truments etc. I set-up the laboratory in one of the rooms of the Hospital for

Infectious Diseases"). From the buboes and blood of patients, Vital Brazil

isolated in pure culture, a coccusbacillus that inoculated in rats, reprodu-

ced the disease. It was thus confirmed that the disease was really bubonic

plague. The outbreak of plague in Santos called the attention of the São

Paulo State Government to the necessity of setting-up a laboratory for the

preparation of the antiplague serum which at that time was prepared in the

Institute Pasteur at Paris in insufficient quantities to attend the require-

ments of the State of São Paulo and other Brazilian States, in view of the

appearance of this disease in the country. Vital Brazil was indicated to set-

up and direct, at a farm near São Paulo, called Butantan, a laboratory for

the preparation of the antiplague and eventually other therapeutic sera.

This laboratory, initially an annex of the Bacteriological Institute, became

independent of this institution in 1901 under the name of Serotherapeutic

Institute. Vital Brazil was nomeated its Director.

In 1901, Vital Brazil published in the São Paulo Medicai Journal his first

papers on snake venoms, some parts of which have herein been quotedA 6

In December of the same year he gave a lecture at the School of Pharmacy

of São Paulo entitled "Snake venom envenomation and its treatment". 8 In

this lecture, he discussed the various resources and methods proposed for

j, | SciELO

VITAL BRAZIL, O. History of the primordia of snake-bite accidenl serotherapy. Mem. Insl. Butantan,

49(11:7-20, 1987.

the treatment of snake bite accidents. Among them, those intended to pre-

vent the absorption of the venom from the site of bite or to destroy it locally

by physical or Chemical means, and those whose aim is to neutralized the

venom already absorbed: the antivenomous serotherapy introduced by

Calmette and based on the discovery by this scientist 15 and Physalix and

Bertrand 25 in 1884 of snake venom immunity. He reported that the antive-

nins are specific since Calmette's "sérum antivenimeux'' did not exert any

neutralizing action whatsoever on the rattlesnake venom while the serum

of animais immunized with this venom, called by him anticrotalic serum,

neutralized it perfectly well. Moreover, Calmette's serum and anticrotalic

serum exerted only a very weak neutralizing action on jararaca venom while

the serum from animais immunized with this venom was very potent in

neutralizing it. The preparation at Butantan of the anticrotalic serum and

antibothropic serum, this last one obtained from animais immunized with

Bothrops jararaca and Bothrops a/fernaíus venoms, and antiophidic serum,

obtained by the mixture of the other two, was announced. The first case of

a snake bite accident treated with an antivenin produced at Butantan was

reported. In 1903 Vital Brazil made a very successful communication to the

Fifth Brazilian Congress of Medicine and Surgery held in Rio de Janeiro, on

snake-bite accident serotherapy and the specificity of the antivenins. At

the opportunity, experiments on pidgeons, rabbits and guinea-pigs showing

the preventive and curative efficacy of the sera prepared at Butantan were

made. In 1903, he also published a paper 9 in the Medicai Journal of São

Paulo in which all his extensive researches on snake venom immunology we¬

re condensed. In this work not only the speficity of the antivenins was

thoroughly demonstrated but also the existence, in certain cases, of a pa-

raspecific action, that is, a serum obtained by the immunization of animais

with a snake venom, can also neutralize, although to a lesser degree, the

venom of a zoological close species exhibiting the same pharmacological

actions. Twenty-one observations of patients bitten by venomous snakes

treated by anticrotalic, antibothropic or antiophidic sera are also presented

in this paper.

At a time of slow Communications, it was necessary to make available

to the rural populations the antivenins prepared at Butantan in order they

might be applied as soon as possible in patients bitten by venomous sna¬

kes, and, at the same time to obtain snakes for venom extraction. For this,

a Service of exchange of the antivenins for snakes was established by Vital

Brazil. Butantan furnished the farmers with the already mentioned "laços"

for catching the snakes without danger of one being bitten and wooden

containers for their transport to the Institute. Thanks to this Service, thou-

sands of venomous and nonvenomous snakes were received annually by

Butantan, stimulating herpetological studies, initiated by Vital Brazil him-

self, set-up by his follower João Florencio Gomes and continued by Afra-

nio do Amaral, Alcides Prado and more recently Alphonse Hoge.

In 1903, three years after the demonstration of antivenin speficity by

Vital Brazil and the start of anticrotalic, antibothropic and antiophidic se¬

rum preparation at Butantan, George Lemb and William Hanna, from the

Medicai and Sanitary Department of Indian Government, published 22 their

first paper on the specificity of the antivenins. They showed in this research

that Calmette's "sérum antivenimeux" did not neutralize the venom from

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49 ( 1 ) 7 - 20 , 1987 .

the elapid Bungarus fasciatus nor those from the viperines Echis carinatus

and Vipera russellii, snakes also responsable for accidents in India. "The

outcome "they wrote", of all these observations is to prove conclusively

that while the serum prepared by Calmette at Lille is of considerable value

as a therapeutic measure in cases of cobra bite if injected sufficiently early

and in sufficient quantity, it is of no value whatever in the treatment of ca¬

ses of bites from Daboia russellii, Bungarus fasciatus or Echis carinatus."

They concluded: "these results... show conclusively that the serum prepa¬

red with a single venom would be specific for the venom of that species,

•that is to say inactive for poisons of other species of other genera". They

published in a second article 23 coming out in 1904, the results of a research

on antivenin specificity using a monovalent serum they prepared with the

venom of cobra. The results of the previous study with Calmette's serum

and a monovalent one obtained by the immunization of horses with tiger

snake (Notechis scutatus) venom from Australia by Tidswell were confir-

med. Besides, they showed that their anticobra venom serum only partially

neutralized the venom of king cobra (Ophiophagus hannah) at that time

classified in the genus Naja (N. bungarus) and whose venom is pharmaco-

logically very similar to that of the commom cobra. "There is no doubt the-

refore that C.V. serum has a certain hindering effect on the action of king

cobra venom in vivo. It cannot, however, be said to have a complete neu-

tralizing effect even when used in large quantities. Further, it is certain that

for praticai therapeutic purposes it would be of no value in cases of bites

from this snake", they stated. Therefore, Lamb and Hanna's researches

confirm entirely those of Vital Brazil on the specificity of the antivenins.

Nowadays mono and polyvalent sera against the main snake venoms of ín¬

dia are prepared at Kasauli and Bombay. 20

Frank Tidswell, from Australia, initiated his studies on snakes venoms

of his country at the end of the last century. In 1902 he reported that the se¬

rum of horses hyperimmunized with the venom of tiger snake did not neu¬

tralize the venom from other Australian snakes. In 1906, an important con-

tribution on Australian snake venoms, snake bite accidents and antivenin

was published by Tidswell. 26 In it he emphasized: "The serum obtained

(antivenin from horses hyperimmunized with the venom of tiger snake)

could validly be regarded only as an antidote for tiger snake venom." He

added: "Unfortunately it could act as an antidate only if the bite had been

inflicted by a tiger snake". Curiosly enough, according to Chippaux and

Goyffon, 20 only monovalent sera against the Australian snake venoms are

produced in this country (possibly due to easy identification of the species

of the snake causing the accident).

Notwithstanding the demonstration of the antivenin specificity by Vital

Brazil, George Lamb and William Hanna as well as by Frank Tidswell, Cal¬

mette modified only partially, in 1907, his opinion on the subject. In his new

concept only two components were responsible for snake venom toxicity

and antivenin specificity: a neurotoxin predominant in venoms from the

Elapidae and a hemorrhagine or proteolytic enzyme in those from the Vipe-

ridae.

In consequence he stated: 17 "Done, chez toutes les espèces de reptiles veni-

meux et peut-etre aussi chez d'autres animaux venimeux (tels que les scor-

pions), il semble que la substance neurotoxique soit unee t toujour neutrali-

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49 ( 11 : 7 - 20 , 1987 .

zable par un sérum antineurotoxique comme celui des animaux vaccinés

contre le venin de Cobra." ("Therefore, in all species of venomous reptiles

and perhaps in other venomous animais also (such as scorpions), it seems

that there is but one neurotoxic substance which is always neutralizable by

an antineurotoxic serum like that from animais immunized against Cobra

venom"). Based on his belief in the unicity of snake neurotoxins he added:

"II" (the serum from animais immunized with cobra venom) "se montre de

même trés suffisamment efficace á 1'égard des venins de Colubridae* et

de Viperidae dont 1'activité neurotoxique peut entraíner la mort" ("It shows

itself very sufficiently effective also in relation to the Colubridae* and Vipe¬

ridae venoms whose neurotoxic action may cause death"). "Mais il ne pos-

sede action empechante", he affirmed further", sur les effects locaux de

1'hemorragine à 1'aquelle certains venins de viperidae tel les Lachesis** —

doivent presque esclusivement leur nocuité" ("But it does not exert any

hindering action on the local effects of the hemorrhagin to which some ve¬

noms of Viperidae — such as the Lachesis* — owe its noxiousness"). Vital

Brazil in an article on antivenomous serotherapy published in 1909 dissen-

ted from Calmette's new ideas on venom specificity. "Infelizmente o gran¬

de número de experiências que temos realizado para elucidar esta ques¬

tão", he wrote, "nos levam a discordar do ilustre professor não só com re¬

lação aos fatos em que se baseia como em relação às conclusões". (Unfor-

tunately the large number of experiments I have done to elucidate this

question, leads me to disagree with the eminent professor not only in rela¬

tion to the facts on which he bases himself but also in relation to his con-

clusions**.) Showing a noteworthy fore-sight in view of the ignorance of

venom chemistry at that time, he added: "A neurotoxina e a hemorragina

são denominações puramente teóricas e não correspondem às substâncias

isoladas e quimicamente puras. Indicam sintomas que se observam no de¬

curso do envenenamento. 0 veneno de nossa cascavel (Crotalus terrificus)

é neurotóxico, segundo a classificação do Professor Calmette, pois tem

ação local muito limitada e mata por ação seletiva no sistema nervoso. A

sua neurotoxina não pode, entretanto, ser identificada à do veneno de Naja

não só pelas diferenças de propriedades como principalmente porque em

doses imunizantes provoca formação de anticorpo diverso" ("The neuroto-

xin and the hemorrhagin are purely theoretical denominations and do not

correspond to chemically pure isolated substances. They merely indicate

symptoms that are observed in the course of the envenomation. The ve¬

nom of our rattlesnake (Crotalus terrificus) is neurotoxic according to Pro¬

fessor Calmette's classification since it has a very limited local action, and

kills by a selective action on the nervous system. Its neurotoxin cannot, ho-

* The elapids were then classified in two subfamilies (Elapmae and Hydrophiinae) of the Colubridae.

** Now Bothrops.

** Vital Brazil always demonstrated great respect and admiration for Calmette's scientific work and a high esteem for

the great French scientist in spite of disagreing entirely with him in regard to the problem of antivenin specificity. Cal¬

mette on the other hand, always showed appreciation for Vital Brazil and his accomplishments. In 1928 by occasion of

a homage rendered to Vital Brazil, he wrote: "L'oeuvre scientifique de Vital Brazil est de tout premier ordre. Ses tra-

vaux sur les venins et sur les serothérapies antivenimeuses ont sauvé des milliers d'existences. Je suis particulierement

hereux de massocier á 1'hommage que vous proposez de lui rendre, L'lnstitut Pasteur de Paris tout entier partage les

sentiments de très haute estime et d'admiration que j'eprouve pour notre illustre collegue et ami”.

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VITAL BRAZIL, 0. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49(11:7-20, 1987.

wever, be identified with that of the Naja ve nom, not only because of diffe-

rences of properties but principally because in immunizing doses, it gives ri-

se to the formation of a distinct antibody.*

Maurice Arthus,the distinguished physiologist from Lausanne, decided

in 1912, to reinvestigate the specificity of the antivenins in view of Calmet-

te's affirmation in 1907 of the unicity of snake venom neurotoxins and he-

morragins and because Pasteur Institute of Lille continued to prepare the

"sérum antivenimeux" using exclusively or predominantly the Naja naja ve¬

nom in the immunization of the horses. His objective: to elucidate whether

venoms from distinct snake species exerting the same pharmacological ac-

tions are equally well neutralizable by an antivenin produced by one of

them. In the first experiments 1 , Arthus used the Lille "sérum antiveni¬

meux" and the venoms from Naja naja (Cobra), Ophiophagus hanna (Ha-

madryas, King Cobra) and Bungarus coeruleus (Krait). After confirming

that from the pharmacological point of view "les venins de Cobra, d'Ha-

madryas et de Krait sont rigoreusement equivalents" (the venoms of Co¬

bra, Hamadryas and Krait are strictly equivalent"), he investigated the neu-

tralizing activity of Calmette's serum in relation to these three venoms. He

found that "pour neutraliser 1 mg de venin de Cobra, il faut 1.4 cc de sérum

antivenimeux; pour neutraliser 1 mg de venin d' Hamadryas il faut environ

20 cc, soit 15 fois plus" (to neutralize 1 mg of Cobra's venom, 1.4 ml of the

"sérum antivenimeux" are needed; to neutralize 1 mg of Hamadryas's ve¬

nom, about 20ml of the sérum are needed or 15 times more"). Arthus fur-

ther verified that Calmette's "sérum antivenimeux" even in a dose of 20 ml

is only capable of hindering the death of the rabbits injected with 2 mg of

Bungarus coeruleus venom (2 mg of the krait venom used equalled in toxi-

city 1 mg of that of Cobra). "De ces experiences, il resulte évidemment que

la substance curarisante des trois venins considerés, la neurotoxine de Cal-

mette, n'est pas une comme le pretend cet auteur: elle varie selon son ori¬

gine zoologique puisqu'elle n'est pas modifiée semblablement à doses ega-

les par le sérum anticobraíque" ("From these experiments, it is evident that

the curarizing substance, Calmette's neurotoxin, in the three venoms is not

one, as it is claimed by this author, since it is not modified in the same way

when applied in equal doses, by the anti-Cobra venom serum"). In a se-

cond group of experiment 2 , Arthus used not only Calmette's "sérum anti¬

venimeux" but also antibothropic and anticrotalic sera from Butantan (fur-

nished by Vital Brazil on Arthus's request) and the venoms from Naja naja,

Bothrops jararaca, Crotalus durissus terrificus, C. adamanteus and

Pseudechis porphyriacus. From the results of both researches, he conclu-

ded: "L'action des sérums antivenimeux est essentiallement specifique, il

s'agit ici de especificité d'origine, de specificité zoologique et non pas de

specificité d'action toxique" ("The action of the antivenins is essentially es-

pecific; it is a especificity of origin, of zoological especificity and not espe-

cificity of toxic action"). He made, however, the following restriction to the

especificity of the antivenins: "Toutefois, cette lois de especificité zoologi¬

que comporte quelques exceptions" ("Nonetheless, this law of zoological

’ Cobra and South American rattlesnake venoms induce neuromuscular blockade. Their neurotoxins are, however,

chemically and pharmacologically, completely differem (see Lee, C.Y., Chemistry and pharmacology of polypeptide

toxins in snake venom, Ann. Rev. Pharmacol., 12:265-281, 1972: Vital Brazil, O.. Neurotoxins from South American

rattle snake venom, J. Formosan Med Assoe , 71:394-400, 1972: Bon, C. et al., Postsynaptic affects of crotoxin and

of its isolated subunits. Eu. J. Biochemen., 99:471 -481, 1979.

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VITAL BRAZIL, 0. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49(11:7-20, 1987.

especificity admits some exceptions") and exemplifying: "Le sérum antico-

braique exerce une action neutralisante, très faible d'ailleurs, sur les vemns

d'Hamàdryas et de Krait, très semblables au venin de Cobra. Les sérums

antibothropique et anticrotalique agissent sur le venin de Crotalus

adamanteus pour en suprimer ou tout au moins pour em atténuer les effets

dépresseurs, mais non pour en neutralizer les effets coagulants in vitro. Le

sérum anticrotalique supprime les effets coagulants in vivo du venin de

Pseudechis porphyriacus, mais il n'agit pas sur ses autre proprietés toxi¬

ques ("the anti-Cobra venom serum exerts a neutralizing action, although

very weak, on the Hamadryas and krait venoms which are very similar to

that of Cobra. The anticrotalic and antibothropic sera suppress or at least

attenuate the depressant effects of Crotalus adamanteus venom but they do

not neutralize its coagulant effect in vitro. The anticrotalic serum suppres-

ses the coagulant effect in vivo of Psudechis porphyriacus venom, but

does not neutralizes its other toxic properties"). At the end of his second

paper 2 , Arthus gave the directives for obtaining really efficient antivenins, di-

rectives already established and followed by Vital Brazil since 1901 in the

preparation of the antivenins at Butantan: "Pour traiter sérotherapeutique-

ment les moursures des serpents venimeux il faut préparer des sérums en

immunisant les chevaux a I'aide du venin dont on se prepose de combatre

les effets chez 1'homme et chez les animaux mordus" ("To treat venomous

snake-bite serotherapeutically, it is necessary that the antivenin be prepa-

red by immunizing the horses with the venom whose effects one wants to

counteract in bitten human beings or in animais").

After carrying out the researches already here referred to, Vital Brazil

kept up his investigations on venoms and antivenins at Butantan. In 1907

he published a paper on the evaluation of the antitoxic activity of

antivenins 10 . The very precise method he developed for this purpose is still

in use nowadays at the Butantan Institute and other Brazilian laboratories.

It was also adopted by the Malbran Institute of Buenos Aires and, with mo-

difications, in other foreign institutions. A very extensive investigation on

the venom of nearly all Brazilian poison snakes was published by Vital Bra¬

zil and Rangel Pestana in 1909 12 ' 13 . The mean quantities of venoms obtai-

ned in thousands of extractions as well as their lethal doses for various la-

boratory animais are reported as well as their coagulant, hemolytic and pro-

teolytic activities in in vitro experiments. The signs and symptoms as well

as the lesions they evoke in dogs were also related. All of Vital BraziLs re¬

searches were characterized by great exactitude. The eminent physiologist

from Argentine, Bernardo Houssay in a paper published in 1923 21 affirmed:

"Nous avons confirmé toujour les recherches si exactes de Vital Brazil"

("We have confirmed always the researches so exact of Vital Brazil"). The

researches of Vital Brazil on venoms and antivenins I have here summari-

zed and those of Carlos Chagas on the American trypanosomiasis (Chaga's

disease) are undoubtly the more important ones done in South America in

the first decades of the present century.

At the end of this address, I want to mention what the distinguished

American pathologist Simon Flexner, director of the Rockefeller Institute

for Medicai Research wrote in 1929 on Vital Brazil scientific work:

"It gives me great pleasure to express to you and your committee the

profund admiration which I have for the scientific work of Dr. Vital Brazil,

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VITAL BRAZIL, 0. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49 ( 11 : 7 - 20 , 1987 .

the founder of the Instituto Butantan in São Paulo. The entire world is in-

debted to Dr. Brazil for his fundamental researches on the venoms and an-

tivenins, and the benefits accruing from the institute he has developed are

felt not only widely in Brazil, but even in distant countries.

I beg to join Dr. BraziTs colleagues in congratulating him on his past

splendid work and in wishing him many more years of fruitful achieve-

ment."

BIBLIOGRAPHIC REFERENCES

1. ARTHUS, M. De la spécíficité des sérums antívenimeux — Sérum anticobraique et ve-

nins d'Hamadryas (Naja bungarus) et de Krait (Bungarus coeruleusl. Arch. int.

Physiol., 7 7:265-284, 1912.

2. ARTHUS, M. De la spécíficité des sérums antívenimeux — Sérums anticobraique, anti-

bothropique et anticrotalique; venins de Lachesis lanceolatus, de Crotalus terrificus et

de Crotalus adamanteus. Arch. int. Physiol., 77:317-338, 1912.

3. BEHRING, E. von & KITASATO, S. Über das zustande-kommer der diphterie-immunitat

und der tetanus-immunitat bei Tieren. Dtsch. med. Wschr., 76:1113-1114, 1890.

4. BERNARD, N. La vie et 1'oevre de Albert Calmette, 1863-1933. Paris, Editions Albin Mi-

chel, 1961.

5. BRAZIL, V. Contribuição ao estudo do veneno ophidico. II. Veneno de algumas espécies

brazileiras ("Contribution to the study of the snake venom. II. The venom of some

Brazilian snake species"). Rev. méd. São Paulo, 4: 296-300, 1901.

6. BRAZIL, V. Contribuição ao estudo do veneno ophidico. III. Tratamento das mordeduras

das cobras ("Contribution to the study of the snake venom. III. Treatment of snake-

bites"). Rev. méd. São Paulo, 4:375-380, 1901.

7. BRAZIL, V. A peste bubônica em Santos ("The bubonic plague in Santos"). Rev. méd.

São Paulo, 2: 343-355, 1899.

8. BRAZIL, V. Do envenenamento ophidico e seu tratamento ("On snake bite envenoma-

tion and its treatment). Conferência realizada em 1,° de dezembro na Escola de Phar¬

macia. Collect. Trab. Instituto Butantan, 7: 31-55, 1918.

9. BRAZIL, V. Tratamento de mordeduras de cobras ("Treatment of snake-bites"). Rev.

méd. São Paulo, 6: 265-278, 1903.

10. BRAZIL, V. Dosagem do valor antitóxico dos soros ("Evaluation of the antitoxic value of

the antivenins"). Rev. méd. São Paulo, 70:457-462, 1907.

11. BRAZIL, V. Serumtherapia antíofídica ("Antivenomous serotherapy"). Rev. méd. São

Paulo, 12: 293-297, 1909

12. BRAZIL, V. & RANGEL PESTANA, B. Nova contribuição ao estudo do envenenamento

ophidico. V. Acção physiologica ("New contribution to the stude of snake venom. I.

Physiological action"). Rev. méd. São Paulo, 72:415-425, 1909.

13. BRAZIL, V. Et RANGEL PESTANA, B. Nova contribuição ao estudo do veneno ophidi¬

co. VI. Acção coagulante. VII. Acção proteolytica ("New contribution to the stude of

snake venom. VI. Coagulation action. VII. Proteolytic action"). Rev. méd. São Paulo,

12: 439-444, 1909.

14. ’CALMETTE, A. Étude experimentale du venin de Naja tripudians ou cobra capei et ex-

posé d'une method de neutralisation de ce venin dans 1'organisme. Ann. Inst. Pas-

teur, 4: 160-183, 1982.

15. CALMETTE, A. L'immunisation artificielle des animaux contre le venin des serpents et la

thérapeutique expérimentale de morsures venimeuses. C.R. Soc. B iol ., 7: 120-124,

1894.

16. CALMETTE, A. Contribution a 1'etude du venin des serpents. Immunisation des ani¬

maux et traitment de l'envenimation. Ann. Inst. Pasteur, 8: 275-291, 1894.

17 CALMETTE, A. Les venins les animaux venimeux et la sérotherapie antivenimeuse. Pa¬

ris, Masson et C ie , 1907.

18. CERQUEIRA LEMOS, F. Contribuição à história do Instituto Bacteriológico 1892-1940.

("Contribution to the history of the Bacteriologic Institute, 1892-1940"). Rev. Inst.

Adolfo Lutz, 74:5-161,1954.

2 3

5 6

11 12 13 14 15

VITAL BRAZIL, O. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan,

49 ( 11 : 7 - 20 , 1987 .

19. CHAGAS, C. Nova tripanozomiaze humana. Estudos sobre a morfologia e o ciclo evolu¬

tivo do Schizotrypanum cruzi n.g. n. sp. agente etiologico de nova entidade mórbida

do homem. Mem. Inst. Osw. Cruz, 7:159-218, 1909.

20. CHIPPAUX, J.P. & GOYFFON, M. Producers of antivenomous sera. Toxicon, 21: 739-

752, 1983.

21. HOUSSAY, B.A. & NEGRETE, J. S peei fiei té de 1'action des serums antivenimeux. C. R.

Soc. Biol., 89: 454-455, 1923.

22. LAMB, G. & HANNA, W. Specificity of antivenomous sera. Scientifics Memoirs by Of-

ficers of the Medicai and Sanitary Department of the Government of India, n.° 5: 1-

14, 1903.

23. LAMB, G. & HANNA, W. Specificity of antivenomous sera. (Second Communication).

Scientific Memoirs by Officers of the Medicai and Sanitary Department of the Go¬

vernment of India, n.° 10: 1-25, 1904.

24. MARTIN, C.Y. The curative value of Calmette's anti-venomous serum in the treatment

of innoculations with poisons of Australian snakes. Intercolonial Med. J. Australasia,

2: 527-540, 1897.

25. PHYSALIX, C. & BERTRAND, G. Surla proprieté antitoxique du sang des animaux vac-

cinés contre le venin de vipère. C. R. Soc. Biol., 7:111-115, 1894.

26. TIDSWELL, F. Snake-bite, snake-venom and antivenine. Researches on Australian Ve-

noms, Department of Public Health, New South Wales. William Applegate Gullick,

Government Printer, Sydney, 1906. p. 4-56.

Recebido e aceito para publicação em 20/11/1986.

Mem. Inst. Butantan

49(1): 21-24, 1987

AVALIAÇÃO “IN VIVO’’ DA VACINA BIVALENTE

CONTRA A GRIPE, PRODUZIDA

NO INSTITUTO BUTANTAN.

Dalva A. Portari MANCINI * *

Cirlene A. VILELA **

Mariangela M. MOREIRA **

RESUMO: Verificou-se o poder de proteção da vacina bivalente contra a

gripe (IB) em camundongos previamente imunizados e posteriormente

infectados com suspensão de vírus influenza adaptado em pulmão de ca¬

mundongo. Foram usados dois grupos de camundongos, corresponden¬

tes às imunizações com vacina pura e diluída a 10' 1 e infectados com

duas cepas do vírus influenza A/SP/1/81 (H, N n ) e A/SP/1/80 (H 3 N 2 ).

A imunidade foi considerada satisfatória, pois resultou sobrevivência

além de 60% (Reed-Muench) para os dois grupos de animais.

PALAVRAS-CHAVE: Vacina contra a gripe, teste de proteção.

INTRODUÇÃO

O problema da gripe é de grande interesse mundial devido a alta infec-

ciosidade da doença e sua incapacidade de produzir imunidade permanen¬

te. Pelo fato de que, de uma determinada epidemia de gripe, ou ainda por

sua principal complicação, a pneumonia, decorram elevados coeficientes

de morbidade e mortalidade, é que a Organização Mundial da Saúde

(OMS) vem desenvolvendo um programa de colaboração internacional pa¬

ra o controle da infecção. Assim, coleta informações epidemiológicas de

toda a parte do mundo, para divulgá-las rapidamente; ainda promove e

coordena pesquisas de laboratório, a fim de descobrir novas variantes de

vírus potencialmente infectantes, fornecendo-as aos laboratórios produto¬

res de vacinas.

A prevenção e o controle da gripe têm sido sustentados, principalmen-

* PqC III — Chefe da Seção de Riquetsias. Serviço de Virologia. Instituto Butantan.

* Estagiárias da Seção de Riquetsias.

2 3

5 6

11 12 13 14 15

MANCINI, D.A.P.; VILELA, C.A.; MOREIRA, M.M. Avaliação "in vivo" da vacina bivalente contra a

gripe, produzida no Instituto Butantan. Mem. Inst. Butantan, 49(1):21-24, 1987

te, por vacinas de diferentes tipos de preparo, com nível de proteção de até

80%, quando utilizadas em casos de epidemias 9 .

A verificação da potência da vacina contra a gripe, geralmente se faz

através de testes "in vitro", porém é importante a avaliação "in vivo" da

proteção e para este teste são utilizados camundongos, hamsters e fu-

rões 4 - 11 - 12 - 13 . A padronização da vacina contra a gripe, através de testes

comparativos "in vitro" e "in vivo", é referida nas investigações de Mc La-

ren e colaboradores 5 .

Estudos da adaptação do vírus influenza em pulmão ou cérebro de ca¬

mundongos adultos ou recém-nascidos, tem colaborado não somente no

controle de vacinas, como também para esclarecimentos da infecciosidade

e da neurovirulência de algumas cepas variantes do vírus

O presente trabalho procura avaliar "in vivo" o poder imunizante da va¬

cina bivalente contra a gripe, composta das cepas A/SP/1/80 (H 3 N 2 ) e

A/SP/1/81 (H,N,) inativadas, produzida no Instituto Butantan e que nor¬

malmente tem sua potência avaliada pela prova de hemaglutinação.

MATERIAL E MÉTODOS

Vírus

Foram empregados na pesquisa, as cepas do vírus influenza, A/SP/1/80

(H 3 N 2 ) e A/SP/1/81 (H,N,), que são mantidas rotineiramente em passa¬

gens sucessivas na cavidade alantóide de ovos embrionados de galinha,

com dez dias de idade. Com o líquido alantóide colhido após 40 horas da

inoculação, foi preparado o inóculo 2 .

Vacina

A purificação e a concentração do vírus foi realizada em supercentrífuga

Sharples, com 50.000 r.p.m., fluxo contínuo, à baixa temperatura ( + 2 a

5°C) 8 . O vírus purificado foi inativado pelo formol na proporção de 1:4000

e ressuspenso em solução tampão fosfato (PBS), de acordo com título he-

maglutinante, de onde resultou a vacina.

Adaptação do vírus em pulmão de camundongo

Para adaptação do vírus foram utilizados dois grupos de 10 camundon¬

gos com peso de 17 a 20 g, para inoculação das cepas variantes

A/SP/1/80 (H 3 N 2 ) e A/SP/1/81 (H,N 3 ) com título hemaglutinante de 128

unidades cada 1 - 10 . Recebeu cada animal, 0,2ml de inóculo, por via nasal

sob leve anestesia com éter. Após cinco dias os camundongos foram rein-

fectados obedecendo a mesma técnica utilizada anteriormente.

Decorrido o prazo de 10 dias, desde o início da primeira infecção, os pul¬

mões dos animais inoculados foram retirados, triturados em gral e ressus-

pensos em solução tampão fosfato (PBS), usando-se Iml para cada pul¬

mão. Em seguida, o material foi centrifugado a 2000 r.p.m. por 30 min. e o

sobrenadante foi testado quanto à infecciosidade, em um total de 20 ca¬

mundongos para cada cepa do vírus, nas diluições 10 1 a 10 4 , com determi¬

nação do título infeccioso (DL 50 ), calculado pelo método Reed-Muench 6 ,

no final do 10.° dia de inoculação. As cepas virais assim adaptadas foram

utilizadas na prova de proteção da vacina.

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10 11 12 13 14 15 16

MANCINI, D.A.P.; VILELA, C.A.; MOREIRA, M.M. Avaliação "in vivo" da vacina bivalente contra a

gripe, produzida no Instituto Butantan. Mem. Inst. Butantan, 49( 1 ):21 -24, 1987

Prova de Proteção "in vivo"

A prova de proteção da vacina, foi realizada em camundongos com pe¬

so de 17 a 20 gramas, inoculando-se por via intradérmica, 0,1ml da vacina

bivalente contra a gripe, de título hemaglutinante de 400 unidades, com¬

posta das duas cepas virais em questão. Utilizou-se vinte camundongos

para cada grupo, isto é, vacina pura e diluída 10 1 . Após sete dias,

inoculou-se a segunda dose. Decorridos 15 dias após a primeira vacinação,

os camundongos foram infectados com as cepas virais adaptadas, sendo

que dez camundongos imunizados com a vacina pura foram infectados

com 100 DL 50 da cepa A/SP/1/80 (H 3 N 2 ) e os outros dez, com 100 DL 50 da

cepa A/SP/1/81 (14,1X1,). Os animais imunizados com a vacina diluída a

10 1 , foram igualmente infectados e todos permaneceram em observação

durante 25 dias. Um controle de 20 camundongos não-imunizados, porém

infectados, foi incluído 5J3 .

RESULTADOS

Os resultados obtidos estão resumidos na tabela.

Verificou-se, através do método de Reed-Muench, a sobrevivência aci¬

ma de 50% para os dois grupos de camundongos e que coincidentemente

apresentaram os mesmos resultados, tanto para vacina pura, como diluída

(10 1 ). Sendo 75% de proteção conferida pela vacina pura, contra 100 DL 50

da cepa A/SP/1/80 (H 3 N 2 ) e a mesma porcentagem de proteção, contra

100 DL 50 da cepa A/SP/1/81 (H,N,).A vacina diluída a 10 1 , conferiu prote¬

ção de 60%, contra as mesmas DL 50 das referidas cepas do vírus. No grupo

controle dos camundongos infectados, mas não imunizados, os níveis de

letalidade foram de 75% para a cepa A/SP/1/81 (H,N,) e de 65% para a

cepa A/SP/1/80 (H 3 N 2 ), o que foi considerado alta infectividade dos vírus

empregados na prova de proteção.

CONCLUSÃO

Pelos resultados obtidos neste trabalho, parece-nos lícito concluir que a

imunização dos camundongos, com vacina bivalente contra a gripe, foi sa¬

tisfatória para o primeiro grupo de camudongos que recebeu vacina pura,

contra 100 DL R0 da cepa do vírus influenza A/SP/1/80 (H 3 N 2 ) e 100 DL

da cepa A/SP/1/81 (H,N,), pois apresentou sobrevivência acima de 50%.

A imunização foi considerada satisfatória, quando este mesmo resultado

foi obtido com o segundo grupo, imunizado com vacina diluída (10 1 ) e

contra as mesmas DL 50 dos vírus influenza.

Podemos concluir ainda, que o teste de proteção "in vivo" é muito

sensível, e importante no processo de avaliação da vacina contra a gripe,

além dos testes "in vitro", tendo em vista que estes nem sempre estão as¬

sociados ao poder imunizante, como observou Lacorte 2 .

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10 11 12 13 14 15

MANCINI, D.A.P ; VILELA, C A.; MOREIRA, M.M. Avaliação "in vivo" da vacina bivalente contra a

gripe, produzida no Instituto Butantan. Mem. Inst. Butantan, 49(11:21-24, 1987.

TABELA

Teste de proteção "in vivo" da vacina bivalente contra gripe

após infecção com vírus influenza

% de Sobrevivência

camundongos

N.°

A (H, N,)

A (H 3 N 2 )

Vac. pura

Vacinados

Vac. 10 1

% de Letalidade

Não Vacina-

A (H, N,)

A(H 3 N 2 )

dos

(controle)

ABSTRACT: The protection of Influenza vaccine was verified in mice

whichhad first been immunised and lateron infected with influenza virus

adapted in mice's lungs. Two groups of mice were used corresponding

immunizations with pure and diluted at 10 1 vaccine and two strains of in¬

fluenza virus A/SP/1/81 (H,N 1 ) and A/SP/1/80 (H 3 N 2 ). The immuniza-

tion was considered satisfatory, because of a survival of above 60%

(Reed Muench) for the two groups of animais.

KEYWORDS: Vaccine against influenza, protection test.

10 .

12 .

13.

REFERÊNCIAS BIBLIOGRÁFICAS

COLLIE, M.H. et al. infection of neonatal and adult mice with non, passage influenza

viruses (Brief report.) Arch. Virol., 65 : 77-81, 1980.

KNIGFIT, C.A., et al. The effect of some Chemicals on purified influenza virus. J. exp.

Med., 79 . 291-300, 1944.

LACORTE, J.G. et al. Dissociação entre as propriedades imunizante e hemaglutinante

do vírus da gripe. Rev. bras. Med., 78(6): 377-84, 1961.

JENNINGS, R. et al. The hamster a model for the study of immunity to influenza virus

infection. Infect. Immun. (session III): 2072-73, 1977.

MC LAREN, C. et al. Comparative antigenicity and immunity of A/USSR/77 influenza

vaccines in normal and primed mice. Infect. Immun., 38(1): 171-77, 1980.

NAKAJIMA, A.S. et al. Neurovirulence of influenza virus in mice. II — Mechanisms of

virulence as studied in a neuroblastoma cell line. Virology, 707: 450-57, 1980.

REED, J. et al. A simple method of stimating fifty percent end points. Amer. J. Hyg.,

27 : 493-97, 1938.

STANLEY, W.M. An evaluation of methods for the concentration and purification of in¬

fluenza virus. J. exp. Med., 79 : 255-66, 1944.

STUART, H.C. The epidemiology and prevention of Influenza. Amer. Scient., 69 : 166-

72, 1981.

SULLIVAN, J. et al. Influenza virus infection in nude mice. J. Infect. Dis., 733(1): 91-4,

1976.

SWEET, C. et al. Differential distribution of virus and histological damage in the lower

respiratory tract of ferrets infected with influenza viruses of differing virulence. J.

gen. Virol., 54 : 103-14, 1981.

TANNOCK, G.A. et al. A clearence test in mice using non adapted viruses to determine

the immunogenicity of influenza strains. Arch. Virol., 70 . 91-101, 1981.

WELLS, M.A. et al. Cytotoxic T-cell and antibody responses to influenza infection of

mice. J. gen. Virol., 43 : 685-90, 1979.

Recebido para publicação em 3/7/1986 e aceito em 8/12/1986.

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10 11 12 13 14 15 16

Mem. Inst. Butantan

49 (1 ):25 33, 1987

SPIDER VENOMS ACTING ON THE

SODIUM CHANNEL *

Oswaldo VITAL BRAZIL **

ABSTRACT: Several toxins — animal, plant and microbial toxins act on

the sodium channel. They may block it, slow down its inactivation or pro-

duce a persistent activation of it. The venom of spiders of the suborder

Labidognatha, Ctenidae family, genus Phoneutria and of the suborder

Orthognata, Dipluridae family, genus Atrax contain toxins that induce

activation and/or slow down of the sodium channel. We have investiga-

ted the mechanism of action and the effects produced by Phoneutria

nigriventer venom at the rat phrenic nerve-diaphragm muscle prepara-

tion. It was found that the venom caused a non-uniform depolarization of

the diaphragm muscle fiber membrane which was abolished by tetrodo-

toxin‘or reduction of the sodium concentration in the bath fluid. The in-

crease in the frequency of the miniature end-plate potentials induced by

the venom was also suppressed by tetrodotoxin. On the other hand, the

duration of action potentials was not increased by the venom. These re-

sults indicated that Phoneutria venom activates the voltage-dependent

sodium channel in muscle and nerve cell membrane. All efects of venom

on the phrenic nerve diaphragm muscle preparation can be explained on

the basis of its action in the sodium channels. Sutherland studied the ac¬

tion of atraxotoxin, the main toxin from Atrax robustus venom, in the

chicken biventer cervicis preparation. It was found that atraxotoxin indu-

ces spontaneous phasic contractions and enhances the response of the

muscle to indirect stimulation. The spontaneous contractions were abo¬

lished by gallamine, succinylcholine, lowered calcium or elevated magne-

sium and by tetrodotoxin. This last observation suggests that atraxotoxin

produces the spontaneous contractions by activating the sodium channel

in nerve terminais.

KEYWORDS: Phoneutria nigriventer venom, Atrax robustus venom, so¬

dium channel.

INTRODUCTION

The discovery of the mechanism of action of venoms and their toxins

presents twofold interest. It permits to clear the pathophysiology of the en-

• Minireview presented in the Spider Venom Satellite Symposium held at Scottsdale, Arizona, in May 23*h, 1986.

* * Department of Pharmacology, Faculty of Medicai Sciences, State University of Campinas, P.O. Box. 6111, Campi¬

nas, São Paulo, Brazil.

2 3

5 6

11 12 13 14 15

VITAL BRAZIL, 0. Spider venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

1987.

venomations they produce and to improve their treatment, and/or to intro-

duce in research new venoms or toxins that may become invaluable tools in

physiological, pharmacological or pathophysiological investigations. The

knowledge of the mechanism of action of such toxins as tetrodotoxin, saxi-

toxin, toxins from scorpions and some sea anemones, veratrum alkaloids,

batrachotoxin and many others that act on the sodium channel has been of

great usefullness from one or both points of view referred to. The study of

the spider venoms acting on the sodium channel is only in its beginning.

Phoneutria nigriventer venom activates the sodium channel in muscle and

motor nerves A® and may also slow down its inactivation in spinal nerve

roots 3 . Sutherland experiments suggest that the main toxin from Atrax

robustus, an Australian Dipluridae spider, activates also the sodium chan¬

nel. Venoms of many other spiders from the Ctenidae or Dipluridae families

probably act likewise activating and/or slowing down the sodium channel.

They evoke signs and symptoms in experimental animais similar to those

produced by Ph. nigriventerven om ,3 .

Phoneutria nigriventer venom

Ph. nigriventer (Ctenidae, Labidognatha), an aggressive wandering so-

litary spider from South America is responsible for most accidents of ara-

neism in center eastern and Southern Brazil. Its neurotoxic venom is very

potent (Table I). The signs and symptoms it evokes in experimental animais

or observed in human accidents are excruciating pain irradiating from the

site of introduction, cramps, tremors, tonic convulsions, paralysis, saliva-

tion, diarrhea, sudoresis, priapism, tachycardia, arrhythmias and visual dis-

turbances 4 ' 5 ' 10 . It does not produce local edema or necrosis, nor blood

coagulation or hemolysis. The venom toxic components are polypeptides

having molecular weight between 4000 and 6000 daltons 10 - 6 .

TABLE 1

50% Lethal Dose to Mice of Some Arthropod Venoms Which Acts

in the Sodium Channel

ARTHROPOD

Route

injection

LD 50

mg/kg

Leiurus quinquestriatus

subc.

0.25 a

Androctonus australis

subc.

0.32 a

Phoneutria nigriventer

subc.

0.67 b

i.v.

0.38 c

Tityus serrulatus

i.v.

0.66 d

Buthacus occitanus

subc.

0.90 a

Centruroides sculpturatus

subc.

1.12 a

a. Zlotkin et al., 1978.

b. Bucherl, 1983.

c. Fontana and Vital Brazil, 1985.

d. Vital Brazil et al., 1973.

í, i SciELO

VITAL BRAZIL, 0. Spider venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

1987.

We have investigated the mode of action of Ph. nigriventer venom at

the isolated rat phrenic nerve-diaphragm preparation 14 ' 7 - 8 . At the concen-

tration of 5 ug/ml, the venom induced a tonic contraction with superimpo-

sed small phasic contractions in unstimulated diaphragms, both effects

being suppressed by d-tubocurarine (Figure 1. I). Therefore, these effects

must be ascribed to a presynaptic action of the venom producing acetyl-

choline release. In indirect stimulated diaphragms, the venom at concen-

trations of 1.0, 5.0 and 25.0 ug/ml produced the following effects: 1 st.) a

dose dependent tonic contraction of short duraction; 2nd.) small sponta-

neous phasic contractions, specially evident at venom concentrations of 5

ug/ml; 3rd.) an increase in twitch tension and delay in twitch relaxation;

4th.) a dose-dependent blockade of neuromuscular transmission at venom

concentrations of 5.0 and 25.0 ug/ml, an effect partially antagonized by

calcium but not by neostigmine or 4-aminopyridine (Figure 1, II, III and IV).

dTc

III JL

JiSg Ca"

felUin.

innminniiiiuiu

FIGURE 1 - Effects of Phoneutria nigriventer venom on muscle contraction. I. Unstimula¬

ted rat diaphragm. A, Addition of the venom, 5 ug/ml, and 14.6 uM d-

tubocurarine to the bath. B, Addition of the venom, 5 ug/ml, to the bath con-

taining a d-tubocurarine-treated rat diaphragm. II, III and IV, Indirectly stimu¬

lated rat diaphragms with maximal shock of 0.1 Hz and 0.2 ms. II, Effects of 1

ug/ml of the venom. A, Before venom addition to the bath; C, 10; D, 20; E,

70; F, 100; G, 140; H, 170 min after venom addition to the bath (paper speed:

A, C, D, E, F, G, 5 cm/s; B. 0.02 cm/s). III, Effect of 25 (a) and 5 ug/ml (b) ve¬

nom. B, C and D, 10, 20 and 40 min after venom addition to the bath. IV, Ef¬

fect of calcium on venom-induced neuromuscular blockade. A, Addition of 25

ug/ml venom to the bath; B, addition of 10 mM CaCI 2 ; C, wash of the prepa¬

ration with Tyrode solution; D, addition of 10 mM CaCI 2 ; E, 50 min after D.

(Fontana & Vital Brazil, 1985).

In curarised directly stimulated diaphragm, the small phasic contrac¬

tions did not occur. Therefore, they are due entirely to acetylcholine relea-

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VITAL BRAZIL, 0. Spider venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

1987.

se. The tonic initial contraction only appeared in the d-tubocurarine treated

diaphragms with the use of 25.0 ug/ml of venom. However, it was signifi-

cantly smaller than that occuring in non-curarized preparations. The increa-

se in twitch tension was smaller in directly stimulated diaphragms than in

the indirectly stimulated ones when 1 and 5 ug/ml of venom were emplo-

yed (Table 2). Twitch relaxation time was smaller in the curarized directly

stimulated diaphragms. In summary, it can be said that at lower concentra-

tions, Phoneutria venom presynaptic action is more important than the

postsynaptic action in the genesis of the effects evoked at the rat phrenic

nerve-diaphragm preparation.

TABLE2

Effect of Phoneutria Nigriventer Venom on

Twitch Tension

Data are reported as the increase in tension (grams) from the baseline to the peak of the

twitches of rat phrenic nerve-diaphragm preparations. The directly stimulated preparations

were blocked with 14.6 uM d-tubocurarine.

Venom

(ug/ml)

Indirectly stimulated rat

diaphragms

Directly stimulated rat

diaphragms

Before venom

After venom

Before venom

After venom

7.3 ± 0.03

11.5 ± 0.08

7.0 ± 0.04

7.5 ± 0.07

8.0 ± 0.03

13.5 ± 0.07

8.7 ± 0.03

10.5 ± 0.06

8.0 ± 0.03

13.5 ± 0.06

8.0 ± 0.03

12.6 ± 0.03

The effect of Ph. nigriventer venom on end-plate potentials (e.p.p.s)

were studied in d-tubocurarine blocked preparations. After venom, a single

shock applied to the phrenic nerve induced a burst of repetitive e.p.p.s. (Fi¬

gure 2). This result shows that the venom causes repetitive firing in the ner¬

ve, originated probably in the pre-terminal part of nerve endings. Repetitive

evoked or spontaneous muscle action potentials were also produced by the

venom (Figure 3). Action potential duration was not increased. This shows

that the venom at the concentrations used does not slow down sodium

channel inativation. The great increase of miniature end plate potential

(m.e.p.p.) frequency induced by the venom was prevented by tetrodotoxin

if added to the bath before venom. When added to the bath after the ve¬

nom had greatly increased the frequency of the m.e.p.p.s., tetrodotoxin

decreaseds it to normal leveis (Figure 4). This effect shows that the increa¬

se in m.e.p.p. frequency is due to depolarization of the membrane of nerve

endings resulting from activation of the sodium channel. The effect of

Phoneutria venon on the muscle resting membrane potential was studied in

preparations blocked by either d-tubocurarine or a-bungarotoxin and in un-

blocked diaphragms. The results did not differ significantly. Depolarization

in five distinct regions of the diaphragm was investigated. It was found that

Ph. nigriventer venom induced an unequal depolarization of the diaphragm

muscle fiber membrane that was blocked by tetrodotoxin. End-plate and

adjacent regions (R2 and RI, Figure 5) were much more depolarized by the

venom than extrajunction regions (R4 and R5, Figure 5) of the diaphragm.

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VITAL BRAZIL, 0. Spider venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

1987.

1.5 mV L

IO msec

FIGURE 2 — Effect of Phoneutria nigriventer venom on the end-plate potential. The rat

phrenic nerve-diaphragm muscle preparation was blocked with 0.73 uM d-

tubocurarine. Nerve stimulation with single shocks. A, Control; B, C and D,

25, 30 and 40 min after addition of 5 ug/ml venom to the bath (Fontana & Vital

Brazil, 1985).

In low sodium (17.2 mM) Tyrode solution the venom did not evoke depola-

rization. These results demonstrate that Ph. nigriventer venom activates

the muscle sodium channel. A similar unequal depolarization of the diaph-

ragm muscle fiber membrane is produced by veratrine 15 46 anc j

crotamine 15 , which activate also the sodium channel. This phenomenon

may be due to a greater density of sodium channels at the end-plate region

of the diaphragm or to a non-uniform distribution of activatable sodium

channels by these toxins along the membrane of the diaphragm muscle fi-

bers. The first hypothesis is favored by the findings that the maximum rate

of rise of the action potential is greater at the end-plate than at extra-

junctional regions 9 ' 12 .2 and that sodium-current density determined with

the use of the loose patch voltage-clamp technique is much higher at re¬

gions immediately adjacent to the end-plate than at regions away from it 5

In conclusion we may say:

1 st.) Ph. nigriventer venom activates the sodium channel in nerve and

muscle cell membrane. Its action in the sodium channel accounts for the

effects produced in the rat phrenic nerve-diaphragm muscle preparation

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10 11 12 13 14 15

VITAL BRAZIL,

1987.

0. Splder venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

30 mV

2 msec

30 mV

5 msec

15 mV

10 msec

FIGURE 3 — Discharges of repetitive action potentials produced by Phoneutria nigriventer

venom. I. Effect of 5 ug/ml venom on action potential evoked by nerve stimu-

lation with a single shock II, Spontaneous discharges of action potentials in

preparations treated with 1.0 (a), 5.0 (b) and 25.0 (c) ug/ml venom (Fontana

Er Vital Brazil, 1985).

- •/•'►VA#

•f; •

*•' "• .<Vi v • i• 'i , ■,, '*,v*

’ ' ' dlf J »•'. 4 i * * * ‘ *

0.7 m V i_,

lOmsec

FIGURE 4 — Effect of Phoneutria nigriventer venom on the miniature end-plate potentials

at the rat phrenic nerve-diaphragm muscle preparation. I, Control; II, III and

IV, 10, 20 and 30 min after the addition of 5 ug/ml venom to the bath; V and

VI, 5 and 15 min after the addition of 3 uM tetrodotoxin to the bath (Fontana

Er Vital Brazil, 1985).

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VITAL BRAZIL, 0. Spider venoms acting on the sodium channel. Mem. Inst. Butantan, 49(11:25-33,

1987.

and may be responsible for all signs and symptoms observed in experimen¬

tal or clinicai envenomation.

2nd.) Ph. nigriventer venom induces bursts of repetitive action poten-

tials which may appear after an evoked action potential or spontaneously.

Spontaneous action potential generation denotes the venom acts also re-

ducing the threshold potential of the excitable cell membrane, that is, the

threshold potential becomes more negative under the action of Ph.

nigriven terven o m.

3rd.) Neurotransmitter release produced by evoked or spontaneous

bursts of repetitive action potentials is the main or unique cause of such ef-

fects as spontaneous phasic or tonic contractions, increase in twitch ten-

sion and delay in twitch relaxation.

-90 Depolanzmg etfect of Phoneutno nigriventer

. - — »A/iÍAnc D. nn/t O aI ♦ k a

-80

-70-

-60-

-50

-45

90 min

FIGURE 5 — Depolarization produced by Phoneutria nigriventer venom at the end-plate (R

2) and an extrajunctional region (R 4) of the rat diaphragm. The preparation

was blocked with d-tubocurarine (14.6 uM). Three concentrations (1.0, 5.0

and 25 ug/ml) of venom were use. Tetrodotoxin (3.0 uM) was added to the

bath 60 min after venom. Each point in the curves is the mean of three experi-

ments.

Atrax robustus Venom

The Orthognatha spiders of the Dipluridae family, genus Atrax occur in

southeastern Australia. A. robustus, a species from the central Coastal re¬

gion of New South Wales and Blue Mountain region to the west is respon¬

sible for severe human accidents. However, fatalities from them are very

low. The signs and symptoms of envenomation are severe local pain lasting

2 3

5 6

11 12 13 14 15

VITAL BRAZIL, 0. Spider venoms acting on the sodlum channel. Mem. Inst. Butantan, 49(1) 25-33

1987.

for hours or even days, nausea and vomiting, abdominal pain, diarrhea, sa-

livation, lacrimation, sweating, hypertension, dyspnea, local and generali-

zed muscle fasciculations. Muscle twitchlng may be prolonged and violent.

Hypotension in some patients preceeds cardiac arrest.

Sutherland 11 studied the action of atraxotoxin, the main toxin from

/\. robustus venom, in the chicken biventer cervicis preparation. He found

that atraxotoxin induces spontaneous phasic contractions and enhances

the response of the muscle to indirect stimulation. The spontaneous con¬

tractions were abolished by gallamine, succinylcholine, lowered calcium or

elevated magnesium. These results show that the contractions were pro-

duced by acetylcholine release caused by discharge of action potentials in

the nerve, very probably originated in the pre-terminal part of the nerve en-

dings. The effect of tetrodotoxin abolishing the spontaneous contractions

suggests but does not prove that atraxotoxin acts in the nerve sodium

channel. Further experiments with the use of electrophysiological techni-

ques are necessary to clarify if atraxotoxin activates or not the sodium

channel.

RESUMO: Várias toxinas — de origem animal, vegetal ou microbiana —

atuam no canal do sódio. Podem bloqueá-lo, retardar sua inativação ou

produzir ativação persistente do mesmo. A peçonha de aranhas da su-

bordem Labidognatha, família Ctenidae, gênero Phoneutria e da subor-

dem Orthognatha, família Dipluridae, gênero Atrax, contém toxinas que

induzem ativação e/ou retardo da inativação do canal do sódio. Investi¬

gamos o mecanismo de ação e os efeitos da peçonha de Phoneutria

nighventer na preparação nervo frênico-diafragma de rato. Verificamos

que a peçonha causa despolarização desigual da membrana das fibras

musculares de diafragma abolida quer pela tetrodotoxina quer pela redu¬

ção da concentração em íons sódio na solução nutritiva. 0 aumento da

frequência dos potenciais da placa terminal em miniatura produzido pela

peçonha também foi abolido pela tetrodotoxina. Por outro lado, a dura¬

ção do potencial de ação, evocado ou espontâneo, não foi alterada pela

peçonha. Estes resultados mostram que a peçonha de Phoneutria ativa o

canal do sódio voltagem-dependente da membrana de fibras nervosas e

musculares. Todos os efeitos produzidos pela peçonha na preparação

nervo frênico-diafragma de rato podem ser explicados como decorrentes

de sua ação no canal do sódio. Sutherland investigou a ação da atraxoto-

xina, a principal toxina da peçonha de Atrax robustus, na preparação

biventer cervicis de pintainhos. Verificou que a atraxotoxina induz con¬

trações fásicas espontâneas e aumenta as respostas do músculo à esti¬

mulação indireta. As contrações espontâneas foram abolidas pela gala-

mina, pela succinilcolina, pela redução na concentração de cálcio ou ele¬

vação da de magnésio na solução nutritiva e pela tetrodotoxina. Esta últi¬

ma observação sugere que a atraxotoxina produza as contrações espon¬

tâneas ativando o canal do sódio nas terminações nervosas.

PALAVRAS-CHAVE: peçonha de Phoneutria nigriventer; peçonha de

Atrax robustus, canal do sódio.

BIBLIOGRAPHIC REFERENCES

1. BEAN, K.G.; CALDWELL, J.H.; CAMPBELL, D.T. Na channels in skeletal muscle con-

centrated near the neuromuscular junction. Nature, 313. 588-590, 1985.

2. BET2, W.J.; CALDWELL, J.H.; KINNAMON, S.C. Increased sodium conductance in

synaptic regions of rat skeletal (muscle fibres). J. Physiol., 352: 189-202, 1984.

SciELO

10 11 12 13 14 15 16

VITAL BRAZIL, 0. Spider venoms acting on the sodium channel

1987.

Mem. Inst. Butantan, 49(11:25-33,

3. BOSTOCK, H. Personal communication.

4. BRAZIL, V. & VELLARD, J. Contribuição ao estudo do veneno das aranhas. Mem. Inst.

Butantan, 2: 5-77, 1925.

5. BRAZIL, V. & VELLARD, J. Contribuição ao estudo do veneno das aranhas. Mem. Inst.

Butantan, 3: 243-299, 1926.

6. ENTWISTLE, J.D.; JOHNSTONE, R.A.W.; MEDZIHRADSZKY, D.; MAY, T.E. Isola-

tion of a puretoxic polypeptide from the venom of the spider Phoneutria nigriventei

and its neurophysiological activity on an insect femur preparation. Toxicon, 20. 1059-

1067, 1982.

7. FONTANA, M.D. & VITAL BRAZIL, O. Pharmacology of Phoneutria nigriventerve nom.

II. Effects on bioelectrical potentials. South African J. Sei., 78: 380-381, 1982.

8. FONTANA, M.D. & VITAL BRAZIL, O. Mode of action of Phoneutria nigriven ter spider

venom at the isolated phrenic nervediaphragm of the rat. BraziUan J. Med. Biol. Res.,

18: 557-565. 1985.

9. NASTUK, W. & ALEXANDER, J.T. Non-homogeneous electrical activity in single mus-

cle fibers. Fed. Proc., 32: 333, 1973.

10. SCHENBERG, S. & PEREIRA LIMA, F. A. Pharmacology of the polypeptides from the

polypeptides from the venom of the spider Phoneutria fera. Mem. Inst. Butantan, 33:

627-638, 1966.

11. SUTHERLAND, S.K. Venoms of Dipluridae. B. Biology and venoms. In: BETTINI, S.

ed. Arthropod Venoms. Berlin, Springer-Verlag, 1978. p. 126-148.

12. THESLEFF, S.; VISKOCIL, F.; WARD, M.R. The action potential in end-plate and ex-

trajunctional regions of rat skeletal muscle. Acta physiol. scand., 91: 196-202, 1974.

13. VELLARD, J. Le venin des araignées. Paris, Masson et C' e , 1936.

14. VITAL BRAZIL, O. & FONTANA, M.D. Pharmacology of Phoneutria nigriventervenom.

I. Effects on the contractile mechanism and neuromuscular transmission. South Afri¬

can J. Sei., 78: 380, 1982.

15. VITAL BRAZIL, O. & FONTANA, M.D. Unequal distribution the sodium channels acti-

vated by veratrine and crotamine at the muscle fiber membrane of rat diaphragm.

Toxicon, 20(1): 80, 1982.

16. VITAL BRAZIL, O. Et FONTANA, M.D. Unequal depolarization of the membrane of the

rat diaphragm muscle fibers caused by veratrine. Pflügers Arch., 404: 45-49, 1982.

Recebido para publicação em 08/10/1986 e aceito em 20/2/1987.

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quina elétrica, em espaço duplo em 3 (três) vias, em papel formato ofício e numerados no ângulo superior direi¬

to.

3. No preparo do original será observada, sempre que possível, a seguinte estrutura: Página de rosto: título do ar¬

tigo, nome(s) do(s) autor(es) e filiação científica. Texto: introdução, material e métodos, resultados, discussão,

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Para livros: autor, título, edição, local de publicação, editor, ano, páginas.

7. BIER, O. Microbiologia e imunologia. 24.ed. São Paulo, Melhoramentos, 1983. 1234p.

Para artigos: autor, título do artigo, título do periódico, volume, página inicial e final, ano.

8. MACHADO, J.C. Sc SILVEIRA F,°, J.F. Obtenção experimental da pancreatite hemorrágica aguda no cão

por veneno escorpiônico. Mera. Inst. Butantan, 40/41: 1-9, 1976/77.

As citações no texto devem ser por números-índices correspondentes às respectivas referências bibliográficas.

Exemplos:

... método derivado de simplificação de armadilha de Disney 1

... segundo vários autores 2 -’ ' 1

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7. BIER, O. Microbiologia c imunologia. 24.ed. São Paulo, Melhoramentos, 1985. 1234p.

Articles

8. MACHADO, J.C. Sc SILVEIRA F.°, J.F. Obtenção experimental da pancreatite hemorrágica aguda no cão

por veneno escorpiônico. Mera. Inst. Butantan, 40/41: 1-9, 1976/77.

Citations in the text should be identificd by thc referente number.

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... método derivado de simplificação de armadilha de Disney 1

... segundo vários autores 2 ' 3 '''

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