A REVIEW OF THE OPSONINS AND BACTERIAL VACCINES.[2]
Reading edition
A REVIEW OF THE OPSONINS AND BACTERIAL VACCINES.[2]
By E. M. Houghton.
As requested by the Therapeutic Gazette, I shall attempt to describe as succinctly as possible the theory of the opsonins and the therapeutic results that have been obtained by the application of the new theory. As the years go by we realize more thoroughly the correctness of the prophetic belief of the “Immortal Pasteur,” that the day would come when it would be possible to eradicate the infectious diseases by vaccination.
We are indebted to Metchnikoff for calling attention to and explaining the rôle of the white blood cells in the defence of the body against bacterial invasion, but the phenomenon of phagocytosis is 142not so simple as it at first appeared, as little by little new facts are developed which amplify our knowledge of the subject.
It was shown by Denys and Leclef, in 1895, that when rabbits were immunized against Streptococcus pyogenes the serum acquired but slight bactericidal properties, but that such serum, when brought in contact with the leucocytes of normal or immunized rabbits, greatly enhanced their phagocytic activity. Conversely no acceleration of phagocytosis was observed when the corpuscles were brought in contact with the serum of a normal animal.
Mennes two years later confirmed this by showing that similar results were obtained from the blood serum of guinea-pigs treated with cultures or toxins of Pneumococci.
A method of measuring the phagocytic activity of the leucocytes was developed by Leishman in 1902. In 1903 Wright and Douglas made an extensive study of the phagocytes, when brought in contact with suspensions of Staphylococcus pyogenes, and were able to show:
(1) Quoting from the original: “We have here conclusive proof that the blood fluids modify the bacteria in a manner which renders them a ready prey to the phagocytes. We may speak of this as an ‘opsonic’ effect (opsono [a Latin verb], I cater for; I prepare victuals for), and we may employ the term ‘opsonins’ to designate the elements in the blood fluids which produce this effect.”
(2) Normal blood serum and plasma possess the same opsonic action upon bacteria.
(3) Serum loses its opsonic action when heated to 60° C. This is spoken of as inactivated serum.
(4) Inactivated serum and physiological salt solution have the same influence upon phagocytosis.
(5) “The opsonic power of the blood fluids disappears gradually on standing,” losing about 50 per cent. of its activity in five or six days.
(6) An anti-opsonic effect is noted when blood serum is digested with typhoid bacilli.
(7) When a condition of immunity is conferred upon patients infected by Staphylococci, by vaccination with heated cultures of Staphylococci, the opsonic action of the patient’s blood serum is greatly augmented.
Neufeld and Rimpau (1904) found in antistreptococcic and pneumococcic 143sera evidence of substances which, while inactive towards leucocytes, possessed very marked opsonic or sensitizing action, as they termed it, toward corresponding cultures of streptococci and pneumococci.
Ross (Lancet, November, 1906) summarizes our knowledge of the opsonins as follows:
“(1) Opsonins act by chemically uniting with the invading bacteria, and so altering them that the leucocytes are able to phagocyte the bacteria and destroy them. It is important to remember that these substances do not stimulate or otherwise affect the leucocytes.
“(2) It is probable that there are present many varieties of opsonins in the blood plasma, each having to do with combating a particular kind of microbic invasion.
“(3) Opsonins have been shown to be distinct from other bacteriotropic substances, such as the bacteriolysins, the agglutinins, and the antitoxins.”
The leucocytes of healthy or diseased persons seem to be equally active when brought in contact with the same serum, hence the amount of opsonins present in the blood of an individual determines, according to the opsonic theory, his susceptibility to bacterial invasion.
TECHNIQUE.
To measure the resistance of the patient to such invasion, or to find out his opsonic index, special technique has been developed, which may be briefly described as follows:
I. EMULSIONS OF BACTERIA.
Twenty-four-hour or younger growths of the rapid-growing bacteria, as Staphylococci, Streptococci, Pneumococci, Gonococci and Colon bacilli, upon inclined agar are washed off with normal saline solution. After the mixture has sedimented, the upper, whitish layer composed of fluid and bacteria is removed with a pipette, and the finer clumps of bacteria precipitated by placing the fluid in a rapidly rotated centrifuge for a few minutes. The supernatant layer, which is still opalescent and is called a bacterial emulsion, should if suitable for work contain the germs in a well-separated condition.
Cultures of tubercle germs are heated, and ground in a mortar with salt solution until the mass is well broken up, and then centrifugated. In case glycerin cultures are used, such as are left in the 144manufacture of Koch’s old tuberculin, the glycerin must be removed by repeated washing with water and finally with 1·5 per cent. salt solution. The washed culture is worked up in a mortar and centrifugated until the clumps are practically all thrown down, and the cloudy layer or emulsion is removed.
The emulsions must be of uniform density. Wright computed the number of germs in a given volume by counting, but McFarland and L’Engle devised an apparatus which is called a nephelometer, consisting essentially of mixtures of BaSO4, put up in sealed tubes, which correspond to solutions containing from 1 to 10 per cent. of BaCl2, which serve as standards. The turbidity of the emulsion is compared in similar layer with the standard tubes of BaSO4. They found that the tube containing “5 per cent. of BaCl2 corresponds to the most useful bacterial suspension.” The permanency of the emulsions varies a good deal. Suspensions of the gonococci should be used at once, staphylococci within two days, etc., while the emulsion of tubercle germs may be employed indefinitely.
II. WASHED WHITE BLOOD-CORPUSCLES.
The finger or lobe of the ear is punctured as in making a blood count, and the blood is allowed to drop directly into normal saline, ·85 per cent. solution, containing about 1 per cent. sodium citrate; this decalcifies the blood and prevents clotting. The corpuscles are completely precipitated in the centrifuge, and then repeatedly washed with ·85 per cent. saline and centrifugated, until all traces of the citrate and serum are removed. After the final precipitation the saline solution is withdrawn, and the thin upper grayish layer of the sediment, the leucocytic cream, consisting for the most part of washed white blood cells, is removed.
III. THE SERUM.
Blood is obtained in the usual way, but is collected in small, bent, glass tubes, which can be readily held in the centrifuge and the serum separated. In obtaining normal serum, care must be exercised in selecting a healthy subject, or, what is better, obtain serum from the mixed blood of several normal persons’ “Pool.”
IV. STAIN.
With the exception of Malta fever and tubercle bacilli, Leishman’s stain, consisting of eosin and methylene-blue, in combination, 145or Jenner’s stain, is employed. Carbol-fuchsin is employed for the tubercle bacilli.
METHOD OF OBTAINING OPSONIC INDEX.
Special capillary pipettes, graduated into equal divisions, are required; then by means of a rubber tube or bulb equal quantities of washed corpuscles, bacterial emulsion, and patient’s serum are taken up, and all blown out upon a glass slide and thoroughly mixed. The mixture is then drawn up in a small pipette, sealed, and placed in an incubator of the ordinary type, or preferably in an opsonic incubator as proposed by Freeman; it is heated to 37° to 40° C. for fifteen minutes. A similar tube is prepared, except that the normal serum or “Pool” is employed instead of the patient’s serum. At the end of fifteen minutes the smears on microscopical slides are made from each tube, fixed, and stained. A good field is selected, one containing many leucocytes, the number of germs counted in the first fifty or hundred observed, and an average per leucocyte determined. If the average in the mixture containing the normal serum is two, we would say that the normal opsonic index is two, and if the mixture containing the patient’s blood serum is one per corpuscle, we would say that his index was one-half, etc.
Wright and his pupils, as the result of numerous observations, classify diseases due to bacterial infections:
(1) “Diseases in which the bacterial process is strictly localized” or “shut off from the lymph and blood circulations.” Furunculosis, lupus, tuberculosis, etc., in fact most chronic infections, belong to this class. “In this class the opsonic index of the blood is persistently below normal, owing to the absence of immunizing stimuli.”
(2) “Diseases in which the bacterial process is but loosely shut off, especially from the lymph circulation.” In these, usually acute, infections immunizing products from the invading bacteria from time to time get into the circulation, and the opsonic index may be normal or above or below normal. Good examples of this class are tuberculous joints, etc.
(3) “Diseases in which the bacterial infection is in the blood stream.” In this class belong acute infectious diseases, septicemia, endocarditis, Malta fever, etc. In these “the opsonic index is generally below normal.”
Wright and Douglas lay down the following general principles for treating cases of bacterial infections:
146(1) “Isolate in pure culture the causative microörganism.”
(2) “Estimate the opsonic power of the patient’s blood to this microörganism.”
(3) “If the opsonic index be at or below normal, prepare and standardize a vaccine from this microörganism.”
(4) “Inoculate the patient with this vaccine with appropriate doses and at proper intervals, as shown by a systematic estimation of the opsonic content of the patient’s blood.”
PREPARATION OF VACCINES.
The vaccines, with the exception of the tubercle vaccine, consist of emulsions of heated cultures of the particular germ producing the infection. Luxuriant cultures of the desired organism are grown upon inclined agar; the growth is then removed with salt solution and a glass rod, and thoroughly emulsified by shaking in a test-tube, in order to get the germs well distributed and the clumps broken up. The number of germs in a given quantity of the emulsion is then determined by comparing the number of germs and red blood-corpuscles in a mixture of one part emulsion, one part freshly drawn blood, and three parts of normal salt solution. If there are ten times as many germs as blood-corpuscles, the bacterial suspension contains approximately 45,000,000 germs per cubic centimeter. The number of germs present will of course vary with the density of the emulsion. The bacterial suspension is heated at the lowest temperature and for the shortest possible time to kill it. The amount of sterilization will differ with different organisms; 60° C. or less maintained for thirty minutes or less is usually sufficient. Cultures from the vaccine are finally made to insure that it is sterile and safe and some preservative added.
Tubercle Vaccine.—This is the “New Tuberculin Koch,” an opalescent fluid, containing the active principle of tuberle bacilli obtained according to the method of Koch.
THERAPEUTIC APPLICATION.
After a proper diagnosis has been made, the patient’s opsonic index taken, and the appropriate vaccine prepared, the patient receives his initial injection of several hundred millions more or less of the heated bacteria, and his opsonic index is watched. A short time after the inoculation is made the opsonic index falls lower than 147it was previous to the injection. This Wright has named the “negative phase.” Sooner or later, from a few hours to several days, the opsonic index rises above the starting-point. This is called the “positive phase.” The amount of the opsonins in the blood remains stationary for a variable length of time, and then diminishes. As soon as their diminution is noted, a second injection of the vaccine is given, which is followed by a negative phase, but less pronounced than before. Soon the positive phase comes on, reaching a higher level than previously. Thus the injections are repeated from time to time according to the opsonic index of the patient’s blood, and the positive phase gradually attains a higher and higher level, until it may be as high as or considerably higher than that of a normal person. In other words, if the vaccinations are properly given, “never during a negative phase,” and as a result the patient’s tissues are stimulated to an increased production of the opsonins, phagocytosis is increased, the invading bacteria are disposed of, and the patient recovers from his infection. The proper handling of patients according to this method requires the greatest attention to details of technique for obtaining the opsonic index, preparing the vaccine, and the administration of the proper dose at the proper time.
It is too early to know the ultimate results that may be expected from the use of the vaccines, but from personal observation, conversation with other workers, and numerous reports, most of which are incomplete, it seems reasonable to believe that for localized bacterial infections much may be accomplished. Especially does this seem probable for acne, furunculosis, sycosis, abscesses, and lupus, adenitis, and other similar tubercular infections.
In incipient pulmonary tuberculosis encouraging results have been obtained where minute doses of new tuberculin, 1 1000 of a milligramme more or less, have been given, which seemingly show that Koch’s original observations were well grounded in fact.
Something has been accomplished in those diseases in which the bacterial infection is in the blood stream.
After a careful consideration of the literature of the entire subject I believe we should recognize the new therapy as an experimental procedure of much promise, but until more extended observations have been made the use of the vaccines should not be looked upon as a settled method of treatment.
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