Br. J. exp. Path. (1976) 57, 431
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR OF RETICULOENDOTHELIAL ACTIVITY E. REGOECZI From the Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8S 4J9 Receivedl for publication, March 12, 1976
Summary.-It is shown that after a single i.v. dose of [1311]-polyvinylpyrrolidone ([131I]-PVP) the total body and plasma radioactivities of rabbits decrease at distinctly
different rates. The difference between these two rates is utilized to calculate the phagocytic rate of [1311]-PVP by reticuloendothelial cells. A number of experimental conditions are reported in which enhanced reticuloendothelial uptake of [1311]-PVP is readily demonstrable. They include the injection of small quantities of heterologous plasma, certain proteolytic fragments of the fibrinogen molecule, the clearance of antigen-antibody complexes, and the acute phase reaction (inflammatory response) as brought about by serum sickness, sterile abscess and vaccination. Based on these observations it is suggested that [1311]-PVP may provide a convenient technique for the long-term monitoring of the activity of reticuloendothelial cells, presumably mainly that of the histiocytes. The pronounced polydispersity of commercially available [1311]-PVP is a serious problem in this respect which can be largely overcome, but not completely abolished, by the screening techniques described herein. Post-mortem analyses of rabbit tissues showed most of the [1311]-PVP to be present in the skin (20%), followed by the liver (14%), bone marrow (10%), muscle (7 0) and kidney (5 0o). Gel filtration studies with [131I]-PVP in the presence and in the absence of plasma proteins failed to demonstrate any association between PVP and the proteins. [13111-PVP kept at physiological pH and 37°C lost less than 5o% of its radioactivity over one month due to spontaneous deiodination.
POLYVINYLPYRROLIDONE (PVP) was first used medically during the Second World War as the colloidal component of the plasma substitute Periston (Hecht and Weese, 1943) to treat haemorrhagic and traumatic shocks. In addition to being a colloid, PVP also binds various mnacromolecules, such as dyes (Bennhold and Schubert, 1944; Bennhold, Ott and Wiech, 1950; Scholtan, 1953) and toxins (Dieckhoff and Kunstler, 1943; Bovet, Couvoisier and Ducrot, 1947; Schubert, 1948) whereby it comes close to what could be termed a " synthetic plasma protein". In more recent years, PVP has been used clinically and experimentally
measure glomerular permeability (Scholtan, 1959; Hecht and Scholtan,
to
1959; Hardwicke et al., 1968; Hulme and Hardwicke, 1968; Arisz et al., 1969), the permeability of the gastrointestinal tract to circulating macromolecules (Gordon, 1959; Jarnum, 1961; Fell et al., 1969), and the capillary transfer of macromolecules from the intravascular to the extravascular space (Vogel and Strocker, 1964). In vitro, PVP proved to be a useful extracellular cryophylactic agent in the preservation of various vertebrate cells (Persidsky and Richards, 1962; Persidsky and Richards, 1963; AshwoodSmith and Warby, 1971; Ashwood-Smith
432
E. REGOECZI
et al., 1972; Damjanovic and Thomas, 1974). PVP is not toxic yet antibody response can be induced with very high mol. wt. PVP (106) in man (Maurer, 1956; Maurer, 1957) and also with lower mol. wt. polymers (24,000-360,000) in mice (Andersson, 1969). This response does not depend on the thymus (Kerbel and Eidinger, 1972; Andersson and Blomgren, 1975). No immune response is observed in rabbits to PVP when challenged with preparations of various average mol. wt. over a wide range (Maurer, 1957). Although it has been realized all along that PVP molecules of < 40,000 daltons are excreted with the urine, the biological fate of the higher polymers remained obscure until 1952 when, in a set of admirably ahead-of-time experiments, Ravin, Seligman and Fine (1952) demonstrated that large PVP molecules are deposited in and stored by the reticuloendothelial cells. Presently available techniques (Stuart, 1970) for the measurement of phagocytic activity in vivo are crude and they do not permit surveillance of the activity of the reticuloendothelial system (RES) over long periods of time, except on a repetitive basis. As a logical extension of the work of Ravin and his colleagues it seemed, therefore, interesting to explore whether labelled PVP could be of use to detect and follow up functional changes in reticuloendothelial activity. MATERIALS AND METHODS
Iodine-labelled PVP.-Two types of preparation were used. The first one was obtained commercially from the Radiochemical Centre (Amersham, England). It had an average mol. wt. of 33,000 (range: 8000-84,000) and was labelled with [131I]. Batches of [l311]_PVP from this source were used for all kinetic studies. The second type of PVP, which had an average mol. wt. of 36,000, was a generous gift from Dr W. Scholtan (Bayer AG, Leverkusen, Germany). To label this material with [1311], a procedure which closely followed that by Briner (1961) was used. In brief, 100 mg PVP was dissolved in 1 ml of 0-2 mol/l H2SO4 at
0°C and placed in a quartz cuvette. Then 0-1 ml sodium nitrite (10% w/v) was added followed by 500 ,uCi Na[131I]. The mixture was irradiated by a u.v. source (254 nm) for 1 h in a stream of cold air after which it was neutralized with 0-2 mol/l KOH and reduced by the addition of 10 mg sodium sulphite in 0-2 ml distilled H20. Unbound radioactivity was removed by anion-exchange chromatography. The labelling efficiency was 20-25%. The product thus obtained was used for studies of the tissue distribution of [1311]-PVP in rabbits and for stability tests (see below). Animals.-Adult New Zealand White and Sandylop rabbits of the Mill Hill strain with an average body weight of 3 kg were used. Theywere kept individually in metabolic cages with a suitable floor arrangement to separate the excreta. Standard pelleted food and drinking water supplemented with 0.005% (w/v) NaI and 0-45% (w/v) NaCl were provided ad libitum. The rats were of the black and white hooded strain. Kinetic studies.-Phagocytic activity was measured as the difference between the slopes of the total body radioactivity and the plasma radioactivity curves of rabbits following the injection i.v. of a single dose of [131I]-PVP (10-20 mg/animal). As the [131I]-PVP contained a significant proportion of molecular sizes prone to rapid renal elimination, the preparations were biologically screened in one of the following ways: (1) screening in a heterologous host. The [1311]-PVP was injected i.v. in a rat and 2-5-3-5 h later the rat was exsanguinated with a heparinized syringe. The blood was centrifuged and 2-2-5 ml portions of the plasma were injected intravenously in the rabbits to be studied. (2) When homologous screening was required, a small rabbit (1 -5-2 kg) was used as the intermediate recipient. Suitable quantities of blood were withdrawn 8-12 h after injecting the [1311]-PVP to obtain 4-6 ml plasma for each of the test animals. (3) In a procedure referred to as autologous screening the [1311]-PVP Was injected in the test animal without using an intermediate host but no radioactivity measurements were undertaken until 51-56 h later. The first values then obtained were taken as the starting values. Behaviour of the [131I]-PVP was followed up for several days (up to a maximum of 15 days) by determining plasma and total body radioactivities at suitable intervals. Plasma samples (0 5 ml + IP5 ml 0 89% NaCl) were set up in duplicate for counting in a Packard model 5212 dual channel automatic gamma counter. Total body [131I] radiation was measured in a ring of 8 Geiger tubes as described by Campbell et al. (1956). Before each count the bladder of the animals was emptied and rinsed with
433
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR
0 899% NaCi solution using a paediatric catheter. The first plasma and total body radioactivity values were taken as 100% and each subsequent value was expressed as a percentage of the corresponding 100% value. Slopes were calculated from a set of experimental points by semilogarithmic regression analysis on computer.
Tissue distribution of [1311]-P_VP.-Three rabbits received [13lI]_PVP i.v. and 8 days later a quantity of blood equivalent to 21% of the calculated total blood volume was removed from each animal. The bleeding, which was aimed at the reduction of [1311]-PVP in transit through the extravascular space (Matthews, 1961), was repeated on the ninth day. On the tenth day the animals were heparinized and exsanguinated from the heart under sodium pentobarbital anaesthesia, approximately 60% of the calculated blood volume being removed at this stage. Following dissection the radioactivity in the organs of interest was determined in the ring counter mentioned above. To make the radiation of organs of different shapes and sizes comparable to each other, [1311] phantom sources of relevant counting geometries were constructed and studied beforehand. Bone marrow, fat and muscles were not collected quantitatively; instead, representative samples of 2-3 g bone marrow, 90-150 g fat and 300400 g muscle were analysed and the total
amounts of these tissues were calculated by reference to the body weight from data in the literature (Nye, 1931; Regoeezi, 1963). Stability tests.-['311]-PVP was tested for spontaneous deiodination and for the constancy of its dispersity as follows. A batch of freshly labelled [131J]-PVP, 4 mg/ml of a 0-125 mol/l sodium phosphate buffer pH 7-38, was divided into two, one half being kept at 5° and the other at 37°C. Both halves were subsampled on Day 0 and then at weekly intervals for a period of 4 weeks. Samples (1 ml) were chromatographed on a Sephadex G-150 column (2-2 cm x 53 cm) which had been calibrated for inorganic iodide as well as for the 19S, 7S and 4S human plasma protein peaks (Schultze and Heremans, 1966). Fractions were counted and the profile of the effluent radioactivity was plotted as a percentage of the load. To gauge dispersity, the chromatogram of the PVP-bound radioactivity was arbitrarily divided into 3 ranges: heavy, medium and light, by reference to the elution volumes of human 19S, 7S and 4S plasma proteins. Thus, the heavy range corresponded to the combined elution volumes of the 19S and 7S protein peaks, the medium range to that of the 48 peak, whereas the light range comprised of polymers smaller than 4S proteins but larger than inorganic iodide (Fig. 1). The relative amount of polymers in each of these ranges was determined by integration.
25 19S 7S 20
4S
-
1.5
P
C4 LU 1.0 2
15-
0
0-0R 10
O 0
E
6n
50
0 0.5
11 100
b'L( 2 150
I O
200
250
300
EFFLUENT (ml) FI-. 1.-Sephadex G-150 chromatogram illustrating the system used to monitor the dispersity of [3][1311]-PVP (*) and the presence of free [1311] in the preparations. The latter is identified through the Na[1 251] (A) added as an internal column marker. The elution profile of the 198, 7S and 4S human plasma protein peaks, obtained by absorbance measurements, is also shown (0). H, M and L are the heavy, medium and light polymer fractions as defined in the Materials and Methods section.
434
E. REGOECZI
[1311]-PVP and plasma protein8.-To see if [1311]-PVP possessed any affinity for plasma proteins, the dispersity of a [1311]-PVP preparation was established by gel filtration as just explained. Then 2 mg [1311]-PVP from the same batch was incubated at 37°C for 1 h with 1 ml fresh human plasma (oxalated) and the mixture was chromatographed on the same gel column. Finally the elution profiles of the radioactivity from both runs were compared. A similar study was also performed after incubating 5 mg [1311]-PVP with 3 mg of the large fragments of trypsin-digested rat fibrinogen (see below). Other techniques.-Human, rabbit and rat fibrinogens of different solubilities were prepared as described before (Regoeczi, 1974). The low-solubility fraction (cryoprofibrin) was obtained from deprothrombinized plasma at 18% saturation with (NH4)2SO4 and normal fibrinogen at 23-8%. Fibrinogen was fragmented with bovine pancreatic trypsin (Worthington, 3 x crystallized) by incubating the protein with the enzyme in 0-125 mol/l phosphate, pH 7 4, at a weight ratio of 155:1 (Mih&lyi and Godfrey, 1963) and at 250C for 6 min. The reaction was stopped by the addition of a 10-fold excess of crystalline soya bean trypsin inhibitor (Worthington) and the large fragments were collected by precipitation with (NH4)2SO4 between 27% and 42% saturations. Occlusion of screened [1251]-PVP by rabbit plasma clots was studied using a technique already described (Regoeezi, 1968). Bovine fibrinopeptides were isolated by a published technique (Regoeczi and Walton, 1967).
TABLE I.-Effect of Temperature on the Spontaneous Deiodination of [1311]-PVP during Storage at pH 7-38 % free radioactivity in stored samples
6torage time , (weeks)
A
at 50 0
0 1 2
0
0
0 2 9 3 9
0 0
3 4
4.5
T/72 9.3h
Z 25
t
-
0
5
10 HOURS
15
FIG. 2.-The initial behaviour of unscreened ['311]-PVP after i.v. administration to a 2-9-kg rabbit. Note the rapid decline in total body radioactivity (A) during the first hours of the experiment and the shape of the plasma radioactivity curve (0) which is not smooth but consists of a set of
exponentials.
20
LABELLED POLYVINYLPYRROLIDONE AS AN IN VI VO INDICATOR
was a severe limitation in its use for the present purpose. However, the quality of the [1311]-PVP could be substantially improved by the various screening procedures already outlined. When rat was used as the intermediate host for periods of 2-5-3 5 h, the initial rapid loss in total body radioactivity of rabbits was reduced to approximately 13% of the dose (Fig. 4). Quite unexpectedly, however, the co-injected rat plasma profoundly affected the phagocytic activity of the rabbit (see below) whereby this approach had to be abandoned. Screening [311I]-PVP for 8-12 h in an intermediate rabbit had a similar reducing effect on polydispersity as the shorter screening in the rat. Nevertheless, the comparatively much larger space available for the distribution of the labelled colloid in the rabbit proved to be a disadvantage of homologous screening. Later work was therefore carried out after performing autologous screening. Sephadex G-150 chromatography of plasma samples obtained from a rabbit at 4 h and 12 h showed marked shifts in the dispersity of [l311]-PVP as the result of screening: 88% of the radioactivity in the 4-h sample represented heavy and the other 12% medium polymers, light polymers being no longer detectable. In the 12-h sample the proportion of heavy polymers was even slightly higher (92%). In both samples all the radioactivity was precipitable by saturation with (NH4)2S04 at 50%.
The phagocytic rate of [131j]-P VP following autologous screening Data relevant to the behaviour of [1311]-PVP in 18 rabbits in which measurements were started 51-56 h after administering the dose are summarized in Table II. It is seen that a striking difference existed between the rate of disappearance of [1311]-PVP from the plasma and that from the body. The differences between these two rates (Ak), calculated individually for each animal, averaged at 0A18 (s.d. + 0 03).
435
From observations with [1311]-PVP screened in heterologous and homologous hosts it is clear that, in analogy to the plasma proteins, PVP does penetrate the extravascular space. However, for the range of heteropolymers used in the present studies, equilibrium between the intra- and extravascular compartments is complete in less than 30 h (e.g. Fig. 4 and 5). It is a reasonable deduction therefore that the divergence between the total body and plasma slopes of [131I]-PVP is a measure for the phagocytosis of PVP by reticuloendothelial cells. During the first 2-4 days following the completion of autologous screening rabbits phagocytize the extracellular PVP at a daily rate of 18 (15-21)%. However, as already pointed out elsewhere (Regoeczi, 1971), in the course of longer studies (10-12 days) this rate may decrease because of changes in the slope of the plasma radioactivity curve. Such changes, which are always from fast to less fast, occur without any obvious reason and are spaced several days apart. Their likely explanation is discussed below. In contrast to the plasma curve, the total body radioactivity curve of [1311]-PVP always remains singleexponential for at least 15 days.
Light polymers and the RES To decide whether the small polymer fractions which are eliminated during biological screening possess any affinity for the reticuloendothelial cells, [1311]PVP was injected in a rabbit and the urine produced over the first 45 min recovered by catheter. The urine was then concentrated to a small volume by pressure dialysis against large volumes of 0.89% NaCl and aliquots of it were injected in other rabbits. The results of one of these studies are summarized in Fig. 3 from which it is evident that most of the light polymers were rapidly re-excreted. Nevertheless, approximately 11-13% of the dose was retained and phagocytized at a relatively high rate (zSk 0.420). =
436
E. REGOECZI
TABLE II. Behaviour of [131I]-PVP in 18 Rabbits Following Autologous Screening* Half-life Radioactivity (days) kt r2t CA Total body 18-0 (±2 3) 0-038 (+0 004) 0-93 (40 04) 0-96 (±0 03) Plasma 3 - 2 ( +0 5) 0-216 (±0 032) 0 97 (+0 02) 0-98 (±0 03) * Values are means with standard deviations in parentheses. t Rate constant of the slope. Coefficient of determination. § Intercept of the slope at zero time with the radioactivity scale, the quantity of radioactivity present at the end of the autologous screening being taken as 1.
[131I]-P VP in imrnmnological episodes The fact that the phagocytosis of PVP could be profoundly influenced by immunological means was first realized through the use of preparations screened in heterologous hosts. As evident from the data in Fig. 4, already small quantities (2 *2 ml) of heterologous plasma greatly stimulated the PVP uptake. The response was of a delayed type. It commenced on the fourth day and lasted for several days thereafter. At its peak, the phagocytic
rate of PVP was increased 4*5-fold. All rabbits receiving ['311]-PVP mixed with heterologous plasma reacted this way, whereas none of those animals receiving the dose in homologous plasma exhibited this phenomenon. The influence of heterologous proteins on [1311]-PVP phagocytosis was further studied by administering human serum albumin (Behringwerke; 15 mg/kg) or bovine IgG (Armour; 8 mg/kg) to rabbits
107
50
T/2- 17.4d
75 -j
25 -
5
T/2 -2.5d
-J
Z lo T/2 -0.62d
Z170
z
0
40
80
HOURS HOURS Fi(e. 3. Total body (A) and plasma (O) radioactivity curves of a rabbit which received [1311]-PVP that had been excreted with the urine during the first 45 min of injection by another rabbit. Half-lives refer to curve sections after 35 h. (See text.)
FIG. 4. Behaviour in a rabbit of [1311]-PVP which had been screened for 2-5 h in a rat and transferred in 2-2 ml heparinized rat plasma. The curves represent total body (A) and plasma (0) radioactivities. The dotted line indicates the expected slope of the plasma curve. Ak before response: 0-237, Ak during maximal response: 1-078.
120
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR
65 h after the completion of [1311]-PVP screening. Human albumin neither provoked an antibody response nor affected the phagocytic rate of PVP. In contrast, antibody response developed 5 days after injecting the bovine IgG and the foreign protein was removed from the circulation at increasing rates in a matter of 50 h. Concurrently, the phagocytic rate of [1311]-PVP was increased 2 2-fold. Although reticuloendothelial clearance of immune complexes always potentiated the phagocytosis of PVP, the two events did not necessarily take place simultaneously. This is concluded from the experiment illustrated in Fig. 5 in which antigen-antibody complexes were formed in vivo by the injection of homologous antiserum to human albumin (kindly provided by Dr J. H. Humphrey, Division of Immunology, N.I.M.R.) slowly followed by an equivalent amount of antigen. It is seen that the prolonged hyperphagocytosis of [1311]-PVP did not commence
437
until some 12 h later, that is, when symptoms of the acute phase reaction (caeruloplasminaemia, hyperfibrinogenaemia, defective fibrin polymerization and lipaemia) began to appear. A repeat experiment in another animal with fourfold antigen excess yielded almost identical results. [1311]-P VP and fibrinogen A plasma protein which, under certain circumstances, can markedly increase the phagocytic rate of [131I]-PVP without the involvement of immunological mechanisms is fibrinogen. Results of a study with a commercial sample of fibrinogen which possessed this stimulating property are summarized in Fig. 6. It will be observed that the injection of the preparation was promptly followed by a 2*6-fold increase in the phagocytic rate of [1311]-PVP. The full effect lasted for approximately 2 days, then the plasma curve of [1311]-PVP began to gradually
100 75 50
= .J
25
-
10
o
7.5
-
T/2 - 2.8d
T
HOURS FIG. 5.-Phagocytosis of [1311]-PVP in a 3.2-kg rabbit before and after injecting homologous antiserum to human serum albumin followed by an equivalent amount (2 .5 mg) of antigen. Time of injection is marked by the thick arrow. For the other symbols see Fig. 4. The [1311]-PVP was screened in a homologous host. Ak before treatment: 0230, Ak during maximal response to treatment: 0728.
438
E. REGOECZI
slow down. When antibodies appeared and the foreign protein was rapidly eliminated from the circulation, once again the [1311]-PVP plasma curve became accelerated. As the potency of further fibrinogen samples from the same source varied widely with respect to stimulating [1311]PVP uptake, it was assumed that some altered form of the fibrinogen molecule, rather than the native protein, was responsible for the observed effect. To test this hypothesis, various homologous and heterologous fibrinogen subfractions were prepared and tested in vivo. Injection of low-solubility fibrinogen (human and rabbit, up to 17 mg/kg), or of fibrinogen preincubated with thrombin (90 mg fibrihogen, 4 'N.I.H. units of thrombin, 3 min) had no detectable effect on the phagocytic rate of [1311]-PVP. Similarly, fibrinogen fractions of normal solubility, and the fibrinopeptides obtained from 350 mg bovine fibrinogen
were ineffective. In contrast, injection of the large fragments of trypsin-digested fibrinogen (7 mg/kg), which themselves had a biological half-life of approximately 3 h, was always followed by a transitory hyperphagocytosis of [1311]-PVP. Also, injection of homologous fibrinogen (15 mg/kg) that underwent partial autolysis as the result of storage in citrate in the unfrozen state for one week increased the phagocytic rate of [131I]-PVP by 52%. In agreement with the effects of the above modifications of the fibrinogen molecule in vitro, abolishment of the circulating fibrinogen in vivo with Arvin (Twyford Laboratories) was followed by a marked increase in the phagocytic rate of [131I]-PVP for approximately 18 h (Fig. 7). The high levels of solubilized Arvin-fibrin found in an earlier study (Regoeezi, Gergely and McFarlane, 1966) are thought to be responsible for this phenomenon.
100 75
50
1 T12 -6
25 CO)
ul
J -I
10
2
7.5
5
2.5
1
280
HOURS FiG. 6. Effect of fibrinogen (human, Kabi grade L, [125I]-labelled, 6-5 mg/kg) on the phagocytosis of [13J1]_PVP in a 3.1-kg rabbit. The [131I]-PVP was subjected to autologous screening for 56 h. Curves denote total body (A) and plasma (0) [131I]-PVP activities and plasma [1251]-fibrinogen ( 0) activity. Ak before injection of the fibrinogen: 0-231, Ak during maximal response to fibrinogen: 0-595.
439
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR ARV
100
CZ)CA31
V
T/2- 14.7d
75
Lu
4
50
-a
25 T2-
T/2-.d
10
7.5
z
E2
5
2.5
0 1
, 40
,
I
80
120
160
HOURS
1
' ' 200 120 160 HOURS F Jo. 7. Effect of Arvin (ARV; twice 2 /lg/kg i.v., spaced 2 h apart) on the phagocytosis of [1311]- PVP in a 3-kg rabbit. The [I311]-PVP was injectedl 56 h prior to the experiment. Curves denote total bo(ty (A) and plasma (0) ['311]-PVP activities and plasma activity of homologous [1251] fibrinogen (0). The lower dliagram shows plasma fibrinogen concentrations (*). Ak before Arvin: 0-191, Ak during the 18 h following Arvini: 0 645.
0
40
80
Fibrinogen was found to possess no affinity for PVP in vitro. This conclusion is borne out of the observation that only 0.1% of the [1311] activity present was occluded by the fibrin mesh when rabbit plasma containing screened [1311]_PVP was converted to serum. The effect of some other treatments on the phagocytic rate of [13 1J]_PVP Intravenous injection of thorium dioxide (2.5 ml of a 25% solution/kg) or heat-aggregated albumin (22 mg/kg) had no effect on the phagocytic rate of [1311]_ PVP. In contrast, injection of unlabelled PVP (25 mg/kg; average mol. wt.: 11,000, range: 6550-30,000) 4 days after the labelled dose reduced the plasma half-life 29
FIG. 8.-Effect of intravenously administered vaccine (V) on the phagocytosis of [I311]-PVP in a 3-6-kg rabbit. The curves represent total body (A) and plasma (0) [1311] radioactivities. Ak before injecting the vaccine: 0-236; Ak during maximal response to vaccine: 0-651. For further details see text.
of [1311]-PVP in a rabbit from 63 to 28 h. The effect lasted for 1 day. Sterile inflammation induced by subcutaneous injection of turpentine (0.5 ml/kg) promptly accelerated the phagocytosis of [1311]-PVP. The effect lasted for at least 4 days and probably much longer, but the experiment had to be terminated prematurely because the intravascular radioactivity reached rather low levels. Observations concerning the effect of a vaccine (Burroughs Wellcome) injected i.v. are summarized in Fig. 8. The dose contained 2 5x 108 S. typhi, 1P25 x 108 of both S. paratyphi A and B, and 2 x 109 V. cholerae. It is seen that on a number of accounts, namely the time gap between challenge and response, as well as the magnitude and duration of the response, the effect of the vaccine on PVP phagocytosis was very similar to that of complexing antigen and antibody
:!00
440
E. REGOECZI
in vivo (Fig. 5). Both types of treatment elicited an acute phase reaction. The tissue distribution of [1311]-PVP The relative radioactivity contents of the organs of 3 rabbits are listed in Table III.
TABLE III1.-Tissue Distribution of the Radioactivity in 3 Rabbits 10 Days after the Intravenous Administration of [1311]-PVP* Tissue Blood Bone marrowt Brain Eye Fatt
Heart Kidney Intestine, large: Intestine, smallt Liver
Lung Musclet Skin Spinal medulla
% total carcase radioactivity 17-5 ± 1-50 10-0 ± 1-19
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR OF RETICULOENDOTHELIAL ACTIVITY E. REGOECZI From the Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8S 4J9 Receivedl for publication, March 12, 1976
Summary.-It is shown that after a single i.v. dose of [1311]-polyvinylpyrrolidone ([131I]-PVP) the total body and plasma radioactivities of rabbits decrease at distinctly
different rates. The difference between these two rates is utilized to calculate the phagocytic rate of [1311]-PVP by reticuloendothelial cells. A number of experimental conditions are reported in which enhanced reticuloendothelial uptake of [1311]-PVP is readily demonstrable. They include the injection of small quantities of heterologous plasma, certain proteolytic fragments of the fibrinogen molecule, the clearance of antigen-antibody complexes, and the acute phase reaction (inflammatory response) as brought about by serum sickness, sterile abscess and vaccination. Based on these observations it is suggested that [1311]-PVP may provide a convenient technique for the long-term monitoring of the activity of reticuloendothelial cells, presumably mainly that of the histiocytes. The pronounced polydispersity of commercially available [1311]-PVP is a serious problem in this respect which can be largely overcome, but not completely abolished, by the screening techniques described herein. Post-mortem analyses of rabbit tissues showed most of the [1311]-PVP to be present in the skin (20%), followed by the liver (14%), bone marrow (10%), muscle (7 0) and kidney (5 0o). Gel filtration studies with [131I]-PVP in the presence and in the absence of plasma proteins failed to demonstrate any association between PVP and the proteins. [13111-PVP kept at physiological pH and 37°C lost less than 5o% of its radioactivity over one month due to spontaneous deiodination.
POLYVINYLPYRROLIDONE (PVP) was first used medically during the Second World War as the colloidal component of the plasma substitute Periston (Hecht and Weese, 1943) to treat haemorrhagic and traumatic shocks. In addition to being a colloid, PVP also binds various mnacromolecules, such as dyes (Bennhold and Schubert, 1944; Bennhold, Ott and Wiech, 1950; Scholtan, 1953) and toxins (Dieckhoff and Kunstler, 1943; Bovet, Couvoisier and Ducrot, 1947; Schubert, 1948) whereby it comes close to what could be termed a " synthetic plasma protein". In more recent years, PVP has been used clinically and experimentally
measure glomerular permeability (Scholtan, 1959; Hecht and Scholtan,
to
1959; Hardwicke et al., 1968; Hulme and Hardwicke, 1968; Arisz et al., 1969), the permeability of the gastrointestinal tract to circulating macromolecules (Gordon, 1959; Jarnum, 1961; Fell et al., 1969), and the capillary transfer of macromolecules from the intravascular to the extravascular space (Vogel and Strocker, 1964). In vitro, PVP proved to be a useful extracellular cryophylactic agent in the preservation of various vertebrate cells (Persidsky and Richards, 1962; Persidsky and Richards, 1963; AshwoodSmith and Warby, 1971; Ashwood-Smith
432
E. REGOECZI
et al., 1972; Damjanovic and Thomas, 1974). PVP is not toxic yet antibody response can be induced with very high mol. wt. PVP (106) in man (Maurer, 1956; Maurer, 1957) and also with lower mol. wt. polymers (24,000-360,000) in mice (Andersson, 1969). This response does not depend on the thymus (Kerbel and Eidinger, 1972; Andersson and Blomgren, 1975). No immune response is observed in rabbits to PVP when challenged with preparations of various average mol. wt. over a wide range (Maurer, 1957). Although it has been realized all along that PVP molecules of < 40,000 daltons are excreted with the urine, the biological fate of the higher polymers remained obscure until 1952 when, in a set of admirably ahead-of-time experiments, Ravin, Seligman and Fine (1952) demonstrated that large PVP molecules are deposited in and stored by the reticuloendothelial cells. Presently available techniques (Stuart, 1970) for the measurement of phagocytic activity in vivo are crude and they do not permit surveillance of the activity of the reticuloendothelial system (RES) over long periods of time, except on a repetitive basis. As a logical extension of the work of Ravin and his colleagues it seemed, therefore, interesting to explore whether labelled PVP could be of use to detect and follow up functional changes in reticuloendothelial activity. MATERIALS AND METHODS
Iodine-labelled PVP.-Two types of preparation were used. The first one was obtained commercially from the Radiochemical Centre (Amersham, England). It had an average mol. wt. of 33,000 (range: 8000-84,000) and was labelled with [131I]. Batches of [l311]_PVP from this source were used for all kinetic studies. The second type of PVP, which had an average mol. wt. of 36,000, was a generous gift from Dr W. Scholtan (Bayer AG, Leverkusen, Germany). To label this material with [1311], a procedure which closely followed that by Briner (1961) was used. In brief, 100 mg PVP was dissolved in 1 ml of 0-2 mol/l H2SO4 at
0°C and placed in a quartz cuvette. Then 0-1 ml sodium nitrite (10% w/v) was added followed by 500 ,uCi Na[131I]. The mixture was irradiated by a u.v. source (254 nm) for 1 h in a stream of cold air after which it was neutralized with 0-2 mol/l KOH and reduced by the addition of 10 mg sodium sulphite in 0-2 ml distilled H20. Unbound radioactivity was removed by anion-exchange chromatography. The labelling efficiency was 20-25%. The product thus obtained was used for studies of the tissue distribution of [1311]-PVP in rabbits and for stability tests (see below). Animals.-Adult New Zealand White and Sandylop rabbits of the Mill Hill strain with an average body weight of 3 kg were used. Theywere kept individually in metabolic cages with a suitable floor arrangement to separate the excreta. Standard pelleted food and drinking water supplemented with 0.005% (w/v) NaI and 0-45% (w/v) NaCl were provided ad libitum. The rats were of the black and white hooded strain. Kinetic studies.-Phagocytic activity was measured as the difference between the slopes of the total body radioactivity and the plasma radioactivity curves of rabbits following the injection i.v. of a single dose of [131I]-PVP (10-20 mg/animal). As the [131I]-PVP contained a significant proportion of molecular sizes prone to rapid renal elimination, the preparations were biologically screened in one of the following ways: (1) screening in a heterologous host. The [1311]-PVP was injected i.v. in a rat and 2-5-3-5 h later the rat was exsanguinated with a heparinized syringe. The blood was centrifuged and 2-2-5 ml portions of the plasma were injected intravenously in the rabbits to be studied. (2) When homologous screening was required, a small rabbit (1 -5-2 kg) was used as the intermediate recipient. Suitable quantities of blood were withdrawn 8-12 h after injecting the [1311]-PVP to obtain 4-6 ml plasma for each of the test animals. (3) In a procedure referred to as autologous screening the [1311]-PVP Was injected in the test animal without using an intermediate host but no radioactivity measurements were undertaken until 51-56 h later. The first values then obtained were taken as the starting values. Behaviour of the [131I]-PVP was followed up for several days (up to a maximum of 15 days) by determining plasma and total body radioactivities at suitable intervals. Plasma samples (0 5 ml + IP5 ml 0 89% NaCl) were set up in duplicate for counting in a Packard model 5212 dual channel automatic gamma counter. Total body [131I] radiation was measured in a ring of 8 Geiger tubes as described by Campbell et al. (1956). Before each count the bladder of the animals was emptied and rinsed with
433
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR
0 899% NaCi solution using a paediatric catheter. The first plasma and total body radioactivity values were taken as 100% and each subsequent value was expressed as a percentage of the corresponding 100% value. Slopes were calculated from a set of experimental points by semilogarithmic regression analysis on computer.
Tissue distribution of [1311]-P_VP.-Three rabbits received [13lI]_PVP i.v. and 8 days later a quantity of blood equivalent to 21% of the calculated total blood volume was removed from each animal. The bleeding, which was aimed at the reduction of [1311]-PVP in transit through the extravascular space (Matthews, 1961), was repeated on the ninth day. On the tenth day the animals were heparinized and exsanguinated from the heart under sodium pentobarbital anaesthesia, approximately 60% of the calculated blood volume being removed at this stage. Following dissection the radioactivity in the organs of interest was determined in the ring counter mentioned above. To make the radiation of organs of different shapes and sizes comparable to each other, [1311] phantom sources of relevant counting geometries were constructed and studied beforehand. Bone marrow, fat and muscles were not collected quantitatively; instead, representative samples of 2-3 g bone marrow, 90-150 g fat and 300400 g muscle were analysed and the total
amounts of these tissues were calculated by reference to the body weight from data in the literature (Nye, 1931; Regoeezi, 1963). Stability tests.-['311]-PVP was tested for spontaneous deiodination and for the constancy of its dispersity as follows. A batch of freshly labelled [131J]-PVP, 4 mg/ml of a 0-125 mol/l sodium phosphate buffer pH 7-38, was divided into two, one half being kept at 5° and the other at 37°C. Both halves were subsampled on Day 0 and then at weekly intervals for a period of 4 weeks. Samples (1 ml) were chromatographed on a Sephadex G-150 column (2-2 cm x 53 cm) which had been calibrated for inorganic iodide as well as for the 19S, 7S and 4S human plasma protein peaks (Schultze and Heremans, 1966). Fractions were counted and the profile of the effluent radioactivity was plotted as a percentage of the load. To gauge dispersity, the chromatogram of the PVP-bound radioactivity was arbitrarily divided into 3 ranges: heavy, medium and light, by reference to the elution volumes of human 19S, 7S and 4S plasma proteins. Thus, the heavy range corresponded to the combined elution volumes of the 19S and 7S protein peaks, the medium range to that of the 48 peak, whereas the light range comprised of polymers smaller than 4S proteins but larger than inorganic iodide (Fig. 1). The relative amount of polymers in each of these ranges was determined by integration.
25 19S 7S 20
4S
-
1.5
P
C4 LU 1.0 2
15-
0
0-0R 10
O 0
E
6n
50
0 0.5
11 100
b'L( 2 150
I O
200
250
300
EFFLUENT (ml) FI-. 1.-Sephadex G-150 chromatogram illustrating the system used to monitor the dispersity of [3][1311]-PVP (*) and the presence of free [1311] in the preparations. The latter is identified through the Na[1 251] (A) added as an internal column marker. The elution profile of the 198, 7S and 4S human plasma protein peaks, obtained by absorbance measurements, is also shown (0). H, M and L are the heavy, medium and light polymer fractions as defined in the Materials and Methods section.
434
E. REGOECZI
[1311]-PVP and plasma protein8.-To see if [1311]-PVP possessed any affinity for plasma proteins, the dispersity of a [1311]-PVP preparation was established by gel filtration as just explained. Then 2 mg [1311]-PVP from the same batch was incubated at 37°C for 1 h with 1 ml fresh human plasma (oxalated) and the mixture was chromatographed on the same gel column. Finally the elution profiles of the radioactivity from both runs were compared. A similar study was also performed after incubating 5 mg [1311]-PVP with 3 mg of the large fragments of trypsin-digested rat fibrinogen (see below). Other techniques.-Human, rabbit and rat fibrinogens of different solubilities were prepared as described before (Regoeczi, 1974). The low-solubility fraction (cryoprofibrin) was obtained from deprothrombinized plasma at 18% saturation with (NH4)2SO4 and normal fibrinogen at 23-8%. Fibrinogen was fragmented with bovine pancreatic trypsin (Worthington, 3 x crystallized) by incubating the protein with the enzyme in 0-125 mol/l phosphate, pH 7 4, at a weight ratio of 155:1 (Mih&lyi and Godfrey, 1963) and at 250C for 6 min. The reaction was stopped by the addition of a 10-fold excess of crystalline soya bean trypsin inhibitor (Worthington) and the large fragments were collected by precipitation with (NH4)2SO4 between 27% and 42% saturations. Occlusion of screened [1251]-PVP by rabbit plasma clots was studied using a technique already described (Regoeezi, 1968). Bovine fibrinopeptides were isolated by a published technique (Regoeczi and Walton, 1967).
TABLE I.-Effect of Temperature on the Spontaneous Deiodination of [1311]-PVP during Storage at pH 7-38 % free radioactivity in stored samples
6torage time , (weeks)
A
at 50 0
0 1 2
0
0
0 2 9 3 9
0 0
3 4
4.5
T/72 9.3h
Z 25
t
-
0
5
10 HOURS
15
FIG. 2.-The initial behaviour of unscreened ['311]-PVP after i.v. administration to a 2-9-kg rabbit. Note the rapid decline in total body radioactivity (A) during the first hours of the experiment and the shape of the plasma radioactivity curve (0) which is not smooth but consists of a set of
exponentials.
20
LABELLED POLYVINYLPYRROLIDONE AS AN IN VI VO INDICATOR
was a severe limitation in its use for the present purpose. However, the quality of the [1311]-PVP could be substantially improved by the various screening procedures already outlined. When rat was used as the intermediate host for periods of 2-5-3 5 h, the initial rapid loss in total body radioactivity of rabbits was reduced to approximately 13% of the dose (Fig. 4). Quite unexpectedly, however, the co-injected rat plasma profoundly affected the phagocytic activity of the rabbit (see below) whereby this approach had to be abandoned. Screening [311I]-PVP for 8-12 h in an intermediate rabbit had a similar reducing effect on polydispersity as the shorter screening in the rat. Nevertheless, the comparatively much larger space available for the distribution of the labelled colloid in the rabbit proved to be a disadvantage of homologous screening. Later work was therefore carried out after performing autologous screening. Sephadex G-150 chromatography of plasma samples obtained from a rabbit at 4 h and 12 h showed marked shifts in the dispersity of [l311]-PVP as the result of screening: 88% of the radioactivity in the 4-h sample represented heavy and the other 12% medium polymers, light polymers being no longer detectable. In the 12-h sample the proportion of heavy polymers was even slightly higher (92%). In both samples all the radioactivity was precipitable by saturation with (NH4)2S04 at 50%.
The phagocytic rate of [131j]-P VP following autologous screening Data relevant to the behaviour of [1311]-PVP in 18 rabbits in which measurements were started 51-56 h after administering the dose are summarized in Table II. It is seen that a striking difference existed between the rate of disappearance of [1311]-PVP from the plasma and that from the body. The differences between these two rates (Ak), calculated individually for each animal, averaged at 0A18 (s.d. + 0 03).
435
From observations with [1311]-PVP screened in heterologous and homologous hosts it is clear that, in analogy to the plasma proteins, PVP does penetrate the extravascular space. However, for the range of heteropolymers used in the present studies, equilibrium between the intra- and extravascular compartments is complete in less than 30 h (e.g. Fig. 4 and 5). It is a reasonable deduction therefore that the divergence between the total body and plasma slopes of [131I]-PVP is a measure for the phagocytosis of PVP by reticuloendothelial cells. During the first 2-4 days following the completion of autologous screening rabbits phagocytize the extracellular PVP at a daily rate of 18 (15-21)%. However, as already pointed out elsewhere (Regoeczi, 1971), in the course of longer studies (10-12 days) this rate may decrease because of changes in the slope of the plasma radioactivity curve. Such changes, which are always from fast to less fast, occur without any obvious reason and are spaced several days apart. Their likely explanation is discussed below. In contrast to the plasma curve, the total body radioactivity curve of [1311]-PVP always remains singleexponential for at least 15 days.
Light polymers and the RES To decide whether the small polymer fractions which are eliminated during biological screening possess any affinity for the reticuloendothelial cells, [1311]PVP was injected in a rabbit and the urine produced over the first 45 min recovered by catheter. The urine was then concentrated to a small volume by pressure dialysis against large volumes of 0.89% NaCl and aliquots of it were injected in other rabbits. The results of one of these studies are summarized in Fig. 3 from which it is evident that most of the light polymers were rapidly re-excreted. Nevertheless, approximately 11-13% of the dose was retained and phagocytized at a relatively high rate (zSk 0.420). =
436
E. REGOECZI
TABLE II. Behaviour of [131I]-PVP in 18 Rabbits Following Autologous Screening* Half-life Radioactivity (days) kt r2t CA Total body 18-0 (±2 3) 0-038 (+0 004) 0-93 (40 04) 0-96 (±0 03) Plasma 3 - 2 ( +0 5) 0-216 (±0 032) 0 97 (+0 02) 0-98 (±0 03) * Values are means with standard deviations in parentheses. t Rate constant of the slope. Coefficient of determination. § Intercept of the slope at zero time with the radioactivity scale, the quantity of radioactivity present at the end of the autologous screening being taken as 1.
[131I]-P VP in imrnmnological episodes The fact that the phagocytosis of PVP could be profoundly influenced by immunological means was first realized through the use of preparations screened in heterologous hosts. As evident from the data in Fig. 4, already small quantities (2 *2 ml) of heterologous plasma greatly stimulated the PVP uptake. The response was of a delayed type. It commenced on the fourth day and lasted for several days thereafter. At its peak, the phagocytic
rate of PVP was increased 4*5-fold. All rabbits receiving ['311]-PVP mixed with heterologous plasma reacted this way, whereas none of those animals receiving the dose in homologous plasma exhibited this phenomenon. The influence of heterologous proteins on [1311]-PVP phagocytosis was further studied by administering human serum albumin (Behringwerke; 15 mg/kg) or bovine IgG (Armour; 8 mg/kg) to rabbits
107
50
T/2- 17.4d
75 -j
25 -
5
T/2 -2.5d
-J
Z lo T/2 -0.62d
Z170
z
0
40
80
HOURS HOURS Fi(e. 3. Total body (A) and plasma (O) radioactivity curves of a rabbit which received [1311]-PVP that had been excreted with the urine during the first 45 min of injection by another rabbit. Half-lives refer to curve sections after 35 h. (See text.)
FIG. 4. Behaviour in a rabbit of [1311]-PVP which had been screened for 2-5 h in a rat and transferred in 2-2 ml heparinized rat plasma. The curves represent total body (A) and plasma (0) radioactivities. The dotted line indicates the expected slope of the plasma curve. Ak before response: 0-237, Ak during maximal response: 1-078.
120
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR
65 h after the completion of [1311]-PVP screening. Human albumin neither provoked an antibody response nor affected the phagocytic rate of PVP. In contrast, antibody response developed 5 days after injecting the bovine IgG and the foreign protein was removed from the circulation at increasing rates in a matter of 50 h. Concurrently, the phagocytic rate of [1311]-PVP was increased 2 2-fold. Although reticuloendothelial clearance of immune complexes always potentiated the phagocytosis of PVP, the two events did not necessarily take place simultaneously. This is concluded from the experiment illustrated in Fig. 5 in which antigen-antibody complexes were formed in vivo by the injection of homologous antiserum to human albumin (kindly provided by Dr J. H. Humphrey, Division of Immunology, N.I.M.R.) slowly followed by an equivalent amount of antigen. It is seen that the prolonged hyperphagocytosis of [1311]-PVP did not commence
437
until some 12 h later, that is, when symptoms of the acute phase reaction (caeruloplasminaemia, hyperfibrinogenaemia, defective fibrin polymerization and lipaemia) began to appear. A repeat experiment in another animal with fourfold antigen excess yielded almost identical results. [1311]-P VP and fibrinogen A plasma protein which, under certain circumstances, can markedly increase the phagocytic rate of [131I]-PVP without the involvement of immunological mechanisms is fibrinogen. Results of a study with a commercial sample of fibrinogen which possessed this stimulating property are summarized in Fig. 6. It will be observed that the injection of the preparation was promptly followed by a 2*6-fold increase in the phagocytic rate of [1311]-PVP. The full effect lasted for approximately 2 days, then the plasma curve of [1311]-PVP began to gradually
100 75 50
= .J
25
-
10
o
7.5
-
T/2 - 2.8d
T
HOURS FIG. 5.-Phagocytosis of [1311]-PVP in a 3.2-kg rabbit before and after injecting homologous antiserum to human serum albumin followed by an equivalent amount (2 .5 mg) of antigen. Time of injection is marked by the thick arrow. For the other symbols see Fig. 4. The [1311]-PVP was screened in a homologous host. Ak before treatment: 0230, Ak during maximal response to treatment: 0728.
438
E. REGOECZI
slow down. When antibodies appeared and the foreign protein was rapidly eliminated from the circulation, once again the [1311]-PVP plasma curve became accelerated. As the potency of further fibrinogen samples from the same source varied widely with respect to stimulating [1311]PVP uptake, it was assumed that some altered form of the fibrinogen molecule, rather than the native protein, was responsible for the observed effect. To test this hypothesis, various homologous and heterologous fibrinogen subfractions were prepared and tested in vivo. Injection of low-solubility fibrinogen (human and rabbit, up to 17 mg/kg), or of fibrinogen preincubated with thrombin (90 mg fibrihogen, 4 'N.I.H. units of thrombin, 3 min) had no detectable effect on the phagocytic rate of [1311]-PVP. Similarly, fibrinogen fractions of normal solubility, and the fibrinopeptides obtained from 350 mg bovine fibrinogen
were ineffective. In contrast, injection of the large fragments of trypsin-digested fibrinogen (7 mg/kg), which themselves had a biological half-life of approximately 3 h, was always followed by a transitory hyperphagocytosis of [1311]-PVP. Also, injection of homologous fibrinogen (15 mg/kg) that underwent partial autolysis as the result of storage in citrate in the unfrozen state for one week increased the phagocytic rate of [131I]-PVP by 52%. In agreement with the effects of the above modifications of the fibrinogen molecule in vitro, abolishment of the circulating fibrinogen in vivo with Arvin (Twyford Laboratories) was followed by a marked increase in the phagocytic rate of [131I]-PVP for approximately 18 h (Fig. 7). The high levels of solubilized Arvin-fibrin found in an earlier study (Regoeezi, Gergely and McFarlane, 1966) are thought to be responsible for this phenomenon.
100 75
50
1 T12 -6
25 CO)
ul
J -I
10
2
7.5
5
2.5
1
280
HOURS FiG. 6. Effect of fibrinogen (human, Kabi grade L, [125I]-labelled, 6-5 mg/kg) on the phagocytosis of [13J1]_PVP in a 3.1-kg rabbit. The [131I]-PVP was subjected to autologous screening for 56 h. Curves denote total body (A) and plasma (0) [131I]-PVP activities and plasma [1251]-fibrinogen ( 0) activity. Ak before injection of the fibrinogen: 0-231, Ak during maximal response to fibrinogen: 0-595.
439
LABELLED POLYVINYLPYRROLIDONE AS AN IN VIVO INDICATOR ARV
100
CZ)CA31
V
T/2- 14.7d
75
Lu
4
50
-a
25 T2-
T/2-.d
10
7.5
z
E2
5
2.5
0 1
, 40
,
I
80
120
160
HOURS
1
' ' 200 120 160 HOURS F Jo. 7. Effect of Arvin (ARV; twice 2 /lg/kg i.v., spaced 2 h apart) on the phagocytosis of [1311]- PVP in a 3-kg rabbit. The [I311]-PVP was injectedl 56 h prior to the experiment. Curves denote total bo(ty (A) and plasma (0) ['311]-PVP activities and plasma activity of homologous [1251] fibrinogen (0). The lower dliagram shows plasma fibrinogen concentrations (*). Ak before Arvin: 0-191, Ak during the 18 h following Arvini: 0 645.
0
40
80
Fibrinogen was found to possess no affinity for PVP in vitro. This conclusion is borne out of the observation that only 0.1% of the [1311] activity present was occluded by the fibrin mesh when rabbit plasma containing screened [1311]_PVP was converted to serum. The effect of some other treatments on the phagocytic rate of [13 1J]_PVP Intravenous injection of thorium dioxide (2.5 ml of a 25% solution/kg) or heat-aggregated albumin (22 mg/kg) had no effect on the phagocytic rate of [1311]_ PVP. In contrast, injection of unlabelled PVP (25 mg/kg; average mol. wt.: 11,000, range: 6550-30,000) 4 days after the labelled dose reduced the plasma half-life 29
FIG. 8.-Effect of intravenously administered vaccine (V) on the phagocytosis of [I311]-PVP in a 3-6-kg rabbit. The curves represent total body (A) and plasma (0) [1311] radioactivities. Ak before injecting the vaccine: 0-236; Ak during maximal response to vaccine: 0-651. For further details see text.
of [1311]-PVP in a rabbit from 63 to 28 h. The effect lasted for 1 day. Sterile inflammation induced by subcutaneous injection of turpentine (0.5 ml/kg) promptly accelerated the phagocytosis of [1311]-PVP. The effect lasted for at least 4 days and probably much longer, but the experiment had to be terminated prematurely because the intravascular radioactivity reached rather low levels. Observations concerning the effect of a vaccine (Burroughs Wellcome) injected i.v. are summarized in Fig. 8. The dose contained 2 5x 108 S. typhi, 1P25 x 108 of both S. paratyphi A and B, and 2 x 109 V. cholerae. It is seen that on a number of accounts, namely the time gap between challenge and response, as well as the magnitude and duration of the response, the effect of the vaccine on PVP phagocytosis was very similar to that of complexing antigen and antibody
:!00
440
E. REGOECZI
in vivo (Fig. 5). Both types of treatment elicited an acute phase reaction. The tissue distribution of [1311]-PVP The relative radioactivity contents of the organs of 3 rabbits are listed in Table III.
TABLE III1.-Tissue Distribution of the Radioactivity in 3 Rabbits 10 Days after the Intravenous Administration of [1311]-PVP* Tissue Blood Bone marrowt Brain Eye Fatt
Heart Kidney Intestine, large: Intestine, smallt Liver
Lung Musclet Skin Spinal medulla
% total carcase radioactivity 17-5 ± 1-50 10-0 ± 1-19
