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A few experiments will serve to show the work of Wright and Douglas.
Mix together blood and staphylococci; incubate fifteen minutes; then make a smear on a slide; stain and examine under the oil-immersion lens. We find that abundant phagocytosis has taken place. Every leucocyte is crowded with cocci; we cannot count them, there are so many in each leucocyte. Thus:
(1) Blood+cocci phagocytosis.
Now mix together washed corpuscles and staphylococci, incubate, and examine as before. We find practically no phagocytosis at all. Practically all the cocci lie outside the leucocytes. Thus:
(2) Washed corpuscles + cocci = 0 phagocytosis.
Now mix washed corpuscles and staphylococci and add blood serum-any human serum will do. Incubate, and examine. We find that phagocytosis has taken place just as when blood was used. Thus:
(3) Washed corpuscles + cocci and serum = phagocytosis.
This shows that there is something in the serum which induces phagocytosis. Does it act upon the leucocytes, stimulating them, or does it act upon the bacteria, altering them, so that they may be taken up? Let us see. If we heat serum at 60° centigrade for ten minutes all this substance is destroyed, and if we mix corpuscles, cocci, and serum thus heated, incubate, and examine we find no phagocytosis. Thus:
(4 Washed corpuscles + cocci + heated serum = 0 phagocytosis.
Now mix cocci and serum together, incubate for fifteen minutes, and heat at 60° centigrade for ten minutes to destroy the protective substance. If we add this mixture to washed corpuscles we find that abundant phagocytosis takes place as in (3). Thus:
Cocci and serum fifteen minutes, then heat at 60° for ten minutes, add washed corpuscles phagocytosis.
They (Wright and Douglas) thus showed that there is a substance in the serum which acts upon bacteria in such a way as to prepare them for phagocytosis. This substance they have called "opsonin," from a Latin word opsono-I prepare food for.
THE NATURE OF OPSONIN.
The most important conclusions follow:
(1) The opsonin exists in the serum and acts upon bacteria and not upon the leucocytes.
(2) Opsonin in normal serum is almost completely destroyed by heating at 60° for ten minutes.
(3) Opsonins possess a high degree of specificity; for example, the blood of a person may contain only one-half the amount of opsonin necessary to counteract a tuberculous infection, and yet have a perfectly normal amount to resist a staphylococcus invasion.
(4) The leucocyte is an invariable factor in phagocytosis. It makes no difference in the amount of phagocytosis whose leucocytes we use: under the influence of a fixed serum the result is the same.
(5) The sera of different persons vary in the amount of opsonin they contain.
(6) Opsonins are distinct bodies differing from the bacteriolysins, agglutinins, antitoxins, et cetera.
The importance of the opsonin over previously discovered antibacterial substances arises from the fact that it can be measured fairly accurately and easily. If we wish to measure the quantity of opsonin to resist staphylococci in a patient suffering, say from furunculosis, which is usually due to an invasion of staphylococci, we need four things:
(1) A few drops of the patient's blood in order to obtain sufficient
(2) A few drops of blood of a normal person in order to have a control serum.
(3) A suspension of staphylococci in salt solution.
(4) Washed leucocytes suspended in salt solution.
In a capillary pipette fitted with a rubber teat equal volumes of corpuscles, the bacterial suspension, and the patient's serum are measured and mixed thoroughly by blowing out on a slide several times. The mixture is then drawn up in the same pipette, the end is then sealed in the flame, and the pipette placed in the incubator. In another pipette, using the same technic, equal parts of washed corpuscles, bacterial suspension, and normal serum are likewise mixed and incubated. At the end of fifteen minutes smears are made from each specimen, fixed and stained. The number of staphylococci per leucocyte is obtained in each case by counting the cocci in the first fifty polymorphonuclear neutrophiles encountered and making an average. The ratio of the average obtained with the test serum to that obtained with the normal serum constitutes the "opsonic index." Thus, if the average for the patient's serum is six cocci per leucocyte and that for the normal serum is ten, the opsonic index to staphylococci for that patient would be 6 10 or 0.6.
The washed corpuscles and the bacterial suspension are the same in each case. They are constant factors. The variable factor is the serum, and you thus compare the test with the normal. The amount of phagocytosis depends upon the quantity of opsonin in the serum. An index of 0.6 means, then, that this patient has six-tenths that amount of opsonin necessary to combat successfully an invasion of staphylococci. If the opsonins are specific it shows nothing of his
resistance to inspection with other microorganisms. To learn this we should have to employ the same method, using the suspension of the germ in question.
From many thousands of determinations on all classes of infection, several general conclusions may be drawn:
(1) In normal healthy individuals the opsonic index varies only within slight limits (from 0.8 to 1.2), and varies only slightly from day to day.
(2) In cases in which the bacterial infection is strictly localized the opsonic index is almost always low, that is, below 0.8. Thus in lupus the index to tubercle bacilli would be found to vary from 0.2 to 0.8 in different cases; or in furunculosis the index to staphylococci would be found to be about 0.6.
(3) In a generalized or systematic inspection the opsonic index may be low or high, but varies markedly from day to day; thus in acute pulmonary tuberculosis the index to tubercle bacilli might be 0.3 one day and two or three days later might be found to be as high as 1.8.
TREATMENT OF BACTERIAL INFECTIONS.
As outlined by Professor Wright, this treatment, briefly stated, consists in inoculating the patient with dead microorganisms of the species causing the infection. Thus in treating a case of furunculosis we use a suspension of staphylococci killed by heating and suspended in normal salt solution. These suspensions of dead microorganisms. Wright calls "vaccines." Thus we have a "colon vaccine" for colon infections; a "tubercle vaccine" for tubercular infections; "gonococcal vaccines"; "pneumococcal vaccines"; et cetera. These vaccines are standardized by counting the number of bacteria by an ingenious method devised by Wright.
Let us see what follows the inoculation of a staphylococcus vaccine in treating a case, say of furunculosis. Shortly after the injection of the vaccine there will be a period of diminished resistance on the part of the body, as shown by a diminution of protective substances in the blood. The particular protective substance that is measured is the opsonin. At this time also the patient will probably be worse clinically. This is called the "negative phase." Succeeding this after a longer or shorter time, depending largely upon the dose of vaccine given, there is an increase in the protective substance, the opsonin, above the original the "positive phase." After a time this tends to wear off and if another injection is given, we get the same train of events, first a fall in the opsonin the "negative phase," followed by a rise-the "positive phase." This is illustrated by the following diagram.
Let us say that the patient has an index of 0.7, and on the second day of the month (see diagram) we inoculate him with a vaccine containing about two hundred million staphylococci. At first we get a
fall in the opsonin to X, the "negative phase." This is followed by a rise to B. After a day or so we give another inoculation, and we get another negative and positive phase. By proper dosage we may be able to raise the index to 1.2 or 1.4-in rare cases even as high as 2.0. As the opsonin falls the patient gets worse, but as it rises he gets better,
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 DAYS OF MONTH
and if we can keep it above 1.0 for a time he will probably get well. The reason that it is necessary to measure the opsonin is this: Should we inoculate a patient during the negative phase, there will be a further fall in the opsonin, or if we give too large a dose the negative phase will be unduly prolonged. This is probably one reason why tuberculin. had such a bad effect in the hands of those who first used it. Too much was given at improper intervals, so that there was a cumulation in the direction of the negative phase. When we give too small a dose, we may get no negative and consequently no positive phase, so we have to give the right dose and at the right time. The initial dose can only be determined by trial, but we start with the dose sufficient to give the least negative phase, and reinoculate only when the postive phase begins to diminish.
I will quote a case reported by Wright and Douglas:
The patient was a man who had suffered from boils almost continuously for four years. His opsonic index was 0.6 to staphylococci on the first examination, and 1.1 on the second. He was inoculated with 2,000,000,000 dead staphylococci. On the day after, the opsonins fell to 0.78, negative phase. From this point there was a steady rise in opsonic power until the original level 1.I was reached a week later. A few days later the index was 1.4, the positive phase. While the
opsonic power was still high another inoculation was given which resulted in, first a negative phase, then a rapid rise for a day or two until the index was 2.0 or twice normal. The clinical result was eminently satisfactory. After several weeks of treatment the boils completely disappeared.
It can be seen, then, that it is of the utmost importance in inoculation to measure the opsonin, for it is only in this way that we can tell the effect of a vaccine and when it is safe to reinoculate. It is believed that the reason why localized infections do not get well of themselves is because insufficient amounts of the bacterial substances are absorbed to cause a rise in opsonic power.
TREATMENT OF TUBERCULOSIS.
For our purpose we divide tuberculous cases into two classes: (1) Those in which the infection is strictly localized, such as lupus, tubercular glands, tuberculosis of subcutaneous tissues, et cetera.
(2) Those which are systemic, as, for example, are most cases of pulmonary tuberculosis-excepting, of course, certain cases of early or quiescent phthisis. The average index to tubercle bacilli in a large series of supposedly healthy persons varies between 0.8 and 1.2. In the class of strictly localized infections the index is almost uniformly low. In cases of lupus the index may be as low as 0.1. This low index is supposed by Wright to antedate the infection and to be the cause and not the result of such infection. In these cases the vaccine used is Koch's new tuberculin, which is in all respects similar to the other bacterial vaccines, consisting as it does of the finely pulverized bodies of tubercle bacilli.
Many cases of lupus, tubercular cystitis, tubercular glands, et cetera, have been reported as cured or benefitted by the inoculation of tuberculin controlled by the estimation of the opsonic index. The train of events following an inoculation is in all respects similar to that described above. There is the negative followed by the positive phase, just as above. Great care must be taken in the treatment with tuberculin not to reinoculate during the negative phase, and the inoculations have to be given at longer intervals than in the case of staphylococcus vaccine, for the increase in opsonic power is maintained over a much longer period. Wright advises as an initial dose 1-1000 milligram of tuberculin powder and subsequent reinoculation with a slightly larger dose only when the positive phase begins to diminish.
The treatment of active pulmonary tuberculosis is very dangerous, for such a person is constantly absorbing tuberculin from the site of infection. It is for this reason that we find the index fluctuating in these cases. One day it may be 0.4, several days later 1.3, and again the next day as low as 0.6. Since we cannot regulate the amount of tuberculin thus absorbed we cannot estimate our dosage. Wright has suggested that such patients be put to bed until the tuberculo-opsonic index gets to one point and stays there-then one may begin treatment with tuberculin.