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`lnt. Arch Allergy 43: 252-268 (1972)
`
`Minimal Molecular Size of Dextran Required to Elicit
`Heterologous Passive Cutaneous Anaphylaxis in Guinea Pigs
`
`W. Richter
`
`The Research Division, Pharmacia AB, Uppsala
`
`Abstract. 1. Isomaltohcxaose and smaller molecular fragments of dextran were found
`incapable of eliciting passive cutaneous anaphylaxis (PCA) in guinea pigs maximally
`sensitized with rabbit antidextrans.
`2. Isomaltodecaose elicited PCA in maximally, but not in submaximally sensitized
`animals. This indicates that monovalency or bivalency is not always expression of a
`physical characteristic of an anaphylaxis elicitor, but may be considered a functional
`property, dependent on the density of cell-fixed antibody populations.
`3. The PCA-eliciting effect of isomaltodecaose could be inhibited by admixture of
`isomaltohcxaose.
`4. The finding that, among the oligosaccharides tested, isomaltodecaose is the smallest
`molecular fragment of dextran capable of eliciting PCA, is in accord with literature data
`on the dimensions of the antidextran-binding site.
`5. PCA titers of 60 samples of rabbit antidextran antisera, estimated in guinea pigs,
`showed a range from 500 to 1,800, with a precipitating antidextran content of 0.8-3.6
`mg/ml in 9 of these sera. The smallest amount of antidextran, detectable by PCA, and
`producing lesions in 50% of sites injected, was found to be 0.02-0.09 //g. An approximately
`linear relationship between diameter of PCA lesions and log dose of antidextran was
`obtained.
`
`Introduction
`
`The dextran-antidextran system shows some physicochemical properties
`differing from those of protein antigen-antibody systems. One of these is the
`simple structure of dextran and the repetitive arrangement of identical anti­
`genic determinants along its threadlike molecules. This permits reduction of
`the molecular size of dextran to a varying extent without loss of immuno­
`logical specificity. In this way, the minimal molecular size required to elicit
`anaphylaxis in guinea pigs sensitized with antidextrans can be studied.
`
`Received: April 19, 1972.
`
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`R ichter
`
`253
`
`In earlier work on hapten inhibition of passive dextran-antidextran
`anaphylaxis, it was found that the minimal molecular size of dextran re­
`quired to elicit systemic anaphylaxis in guinea pigs sensitized with large
`doses of rabbit antidextrans corresponded to a weight average molecular
`weight (Mw) of about 3,000. Larger molecular size dextran was required to
`elicit anaphylaxis in animals sensitized with smaller amounts of antidextrans
`[Richter, 1971 b].
`To study this problem further, passive cutaneous anaphylaxis (PCA) has
`been chosen as a model. PCA is more sensitive, allows better quantitation
`and requires less animals than systemic passive anaphylaxis. A series of
`isomaltose oligosaccharides (10) of increasing molecular size and some
`dextran fractions have therefore been tested as to their capability to elicit
`PCA in animals sensitized with varying amounts of antidextrans. The
`molecular properties of antigens or haptens, necessary for the elicitation of
`anaphylaxis, will also be discussed.
`
`Materials and Methods
`
`Animals. Albino guinea pigs of both sexes, 250-350 g, and albino rabbits of the ‘vit
`landras’ strain, 3-5 kg, were used.
`Antidextran antisera. Antisera were raised in rabbits by repeated immunization with
`soluble dextran-protcin conjugates, emulsified in complete Freund’s adjuvant, as described
`earlier [Richter, 1970, 1971a, b]. Preparation of the dextran-protein conjugates by the
`cyanogen bromide method has been described recently [Richter and Kagedal, 1972],
`Passive cutaneous anaphylaxis. The method was used as introduced and described by
`Ovary [1952, 1958], with some minor modifications [Richter, 1970]. A time of 3 h was
`allowed between passive sensitization with 0.1-ml volumes of suitably diluted rabbit anti­
`dextran antisera and i.v. challenge. Eight intradermal sites of abdominal skin were
`sensitized in each animal. Before clipping of hair and intradermal sensitization, animals
`were slightly anesthetized with 25 mg/kg of nembutal sodium i.p. Challenging injections
`were given in volumes of 0.5-1.0 ml into an ear vein using a thin polyethylene catheter
`with attached, cut off intradermal injection needle [see also Hint and R ichter, 1958].
`Prior to inserting the needle, 0.02 ml of 2% lidocain solution are injected at the base of the
`ear, to produce vcnular dilation and analgesia. The injection of the dextran or oligo­
`saccharide solution was immediately followed by a dose of 10 mg/kg of Evan’s blue,
`dissolved in saline. 30 min after challenge, animals were sacrificed, the skin reflected and
`excised and the size of PCA lesions measured. Colour intensity of lesions was assessed by
`inspection, using an arbitrary scale of 0, 1, 2, 3, 4.
`PCA titer. Serial twofold dilutions of rabbit antidextran antiserum were injected into
`3-4 sites in 3-4 animals per dilution step. PCA lesions with diameters smaller and larger
`than 10 mm were produced and the PCA titer was defined as the reciprocal of the extra
`
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`254
`
`Richter
`
`Table 1. Examples of PCA liter estimations of some rabbit antidextran antisera showing
`relationship between dilutions of antisera of varying strength used for sensitization and
`size of PCA lesions. Each figure in the table represents the average diameter (mm) of
`PCA lesions, calculated from 6-8 spots in a group of 3-4 guinea pigs. Figures in brackets
`are serum dilutions. Serial twofold serum dilutions have always been employed. For
`challenge, dextran M„ 71.000 in a dose of 0.15 mg/kg i.v. was used
`
`Column
`
`A
`
`Dilution step No. 1
`
`B
`
`2
`
`C
`
`3
`
`D
`
`4
`
`E
`
`5
`
`F
`
`PCA
`titer
`
`Antiserum A
`Antiserum B
`Antiserum C
`Antiserum D
`Antiserum E
`
`21 (1:160)
`17(1:10)
`16(1:40)
`19(1:160)
`-
`
`13 (1:320)
`16(1:20)
`15 (1:80)
`15 (1:320)
`14(1:640)
`
`6(1:1.280)
`10(1:640)
`3(1:80)
`11 (1:40)
`6(1:320)
`12(1:160)
`8 (1:640)
`4(1:1,280)
`8 (1:1,280) 6(1:2,560)
`
`2 (1:2,560)
`
`640
`40
`200
`600
`3 (1:5,120) 1,000
`
`Average diameter
`of lesions, mm
`Total number of
`lesions
`
`18
`
`28
`
`15
`
`38
`
`10
`
`38
`
`5
`
`40
`
`3
`
`8
`
`To make results from PCA titer estimations of antisera of different strength compar­
`able, the following procedure was adopted. For every titration, average lesion diameters
`from each dilution step were calculated and grouped according to size. Thereafter, PCA
`lesions with average diameters nearest to 10 mm were allotted to column C above. Finally,
`lesions with average smaller or larger diameters from adjacent dilution steps were allotted
`to columns D and E, or B and A, respectively.
`
`polated dilution of antiserum, producing lesions with a diameter nearest to 10 mm, on
`challenge with a dose of 150 /tg/kg of dextran Mw 71,000.
`Dextran Fractions and Oligosaccharides o f the Isomaitosc Scries. Dextran fractions.
`After hydrolysis of native dextran produced by Leuconostoc mesenteroides NRRL B 512,
`fractions were prepared cither by repeated fractionation with ethanol or by preparative gel
`filtration on Sephadex columns [Granath, 1964]. M,v was determined by light scattering
`and number average molecular weight (M n) by end group analysis [Isbell et al., 1953].
`Molecular weights of the fractions used are given in tables V, VI and VII.
`Oligosaccharides. These were prepared from a hydrolysate of dextran B 512 by carbon
`column chromatography. The purity was checked by paper chromatography, using
`Whatman No. I paper, and ethyl acetate-pyridine-water (10-5-5 v/v) as eluent. Each oligo­
`saccharide produced a single spot. The spots were visualized by the silver nitrate sodium
`hydroxide reagent. IO composed of up to 12 glucose residues were prepared.
`
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`Molecular Size of Dextran Eliciting PCA in Guinea Pigs
`
`255
`
`Table II. Relation between amount of sensitizing antibody per skin site, percentage of
`sensitized sites giving PCA lesions upon antigen challenge, and average size of PCA lesions
`(figures in brackets are approximate amounts of antidcxtran [//g] injected per site, re­
`presenting average from 9 antisera titrations). Data arc taken from 60 titer estimations of
`PCA-reactive rabbit antidcxtran antisera in guinea pigs. For i.v. challenge, dcxtran M,v
`71,000 was used in a dose of 0.15 mg/kg
`
`Column
`
`A
`
`B
`
`C
`
`D
`
`E
`
`Serum dilution step No.
`Percentage of sites
`sensitized resulting
`in PCA lesions1
`Total number of
`PCA lesions
`Average diameter of lesion,
`mm (m eaniSD)
`Average area per lesion
`(diameter, mm-)
`
`1 (0.70)
`
`2 (0.35)
`
`3 (0.18)
`
`4 (0.09)
`
`5 (0.05)
`
`100
`
`210
`
`100
`
`371
`
`98
`
`420
`
`70
`
`360
`
`52
`
`84
`
`18 ± 1.8
`
`15 ±2.4
`
`12 ± 2.4
`
`6± 1.9
`
`3± 1.5
`
`324
`
`225
`
`144
`
`36
`
`9
`
`' Upon i.v. challenge with dextran M* 71,000, 0.15 mg/kg.
`Serial twofold dilutions of antisera were used and 0.1-ml volumes were injected per
`skin site. For each dilution step, 6-8 sites in 3-4 guinea pigs were injected. To make
`titration results of antisera of different strength comparable, PCA lesions from each dilu­
`tion step were grouped according to size. At first, lesions of average diameters nearest to
`10 mm were allotted to column C above. Thereafter, lesions with average smaller or larger
`diameters from adjacent dilution steps were allotted to columns D and E, or B and A,
`respectively.
`
`Results
`
`Data from a total of 60 PCA titer estimations of rabbit antidextran
`antisera in guinea pigs are presented in tables I—III. In these experiments-
`animals were challenged i.v. with dextran Mw 71,000. using a dose of
`150 /tg/kg. The effective challenging dose range of dextran has been de‘
`termined earlier [R ichter, 1970], using a large dose of sensitizing antif
`dextran. Comparable experiments with a second rabbit antiserum o-
`known antidextran content (ODX3 = 1.3 mg/ml) gave similar results and are
`summarized in table IV. It is evident that a dose of 150 /tg/kg must be con­
`sidered large, exceeding the minimal dose necessary to elicit PCA in animals
`sensitized with a large dose of antidextran about 100 times. A tenfold reduc-
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`256
`
`R ichter
`
`Table III. PCA titer estimations of some rabbit antidextran antisera. Relation between
`amount of antidextran injected per skin site and size of PCA lesion upon challenge with
`a large antigen dose. Code designations of antisera within the first series of brackets.
`Figures in the second series of brackets are serum dilutions corresponding to serum
`dilution step No. 1. Challenge with dextran M« 71.000 i.v.. 0.15 mg/kg
`
`Column
`
`Scrum dilution step No.
`
`/ig of precipitating antidextran
`injected per site
`Titration No. 1 (ODX3) (1 200)
`(1 100)
`Titration No. 2 (KPX)
`Titration No. 3 (HDX1) (1 500)
`(1 125)
`Titration No. 4 (PD4A)
`(1 300)
`Titration No. 5 (PD4B)
`(1 300)
`Titration No. 6 (K.P8)
`(1 300)
`Titration No. 7 (KP7)
`Titration No. 8 (KP6)
`(1 150)
`Titration No. 9 (KP5)
`(1 150)
`
`Average
`
`Average diameter of lesion. mm
`
`A
`
`1
`
`1.30
`0.92
`0.72
`0.96
`0.40
`0.32
`0.40
`0.80
`0.52
`
`0.70
`
`21
`
`B
`
`2
`
`0.65
`0.46
`0.36
`0.48
`0.20
`0.16
`0.20
`0.40
`0.26
`
`0.35
`
`17
`
`C
`
`3
`
`0.33
`0.23
`0.18
`0.24
`0.10
`0.08
`0.10
`0.20
`0.13
`
`0.18
`
`12
`
`D
`
`4
`
`0.16
`0.12
`0.09
`0.12
`0.05
`0.04
`0.05
`0.10
`0.06
`
`0.09
`
`3
`
`E
`
`5
`
`0.08
`0.06
`0.05
`0.06
`0.03
`0.02
`0.03
`0.05
`0.03
`
`0.05
`
`2
`
`The amount of precipitating antidextran in the sera above (ODX3 to KP5), estimated
`either by quantitative precipitation or reversed single radial immunodiffusion, was as
`follows (mg/ml): ODX3=1.3, KPX = 0.9, HDX1=3.6. PD4A=1.2, PD4B=I.2, KP8 =
`0.9, KP7 = 1.2, KP6 = 1.2, KP5=0.8. PCA titers for the same sera were as follows: 600,
`600, 1,500, 800, 1,200, 1,800, 1,200, 800, 600. * l
`
`tion of antigen dose to 15 /rg/kg still produces lesions in 100% of sites
`sensitized, the average area per lesion being only moderately smaller. A
`further tenfold reduction of dextran dose to 1.5 //g/kg still produced lesions
`in 25-50% of sites sensitized, and the average area per lesion was about
`lh t0 Va of that produced by 150 ¿tg/kg of dextran.
`The PCA titrations of the antidextran antisera shown in table 1 exemplify
`how antisera of different strength were compared. By grouping equipotent
`dilutions of different antisera into five PCA size categories, a large number of
`titrations became comparable. It is seen, that the average PCA lesion dia­
`meters from 5 to 9 titrations (tab. I, III) do not deviate markedly from those
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`Molecular Size of Dextran Eliciting PCA in Guinea Pigs
`
`257
`
`Table IV. Lowest i.v. challenging dose of dextran Mw 70,000 required to elicit hetero­
`logous PCA in guinea pigs sensitized with a large dose of rabbit antidextran
`
`Code of antiserum Challenging dose Number of Average spot % of sites injected
`(dilution used)
`of dextran, fig/kg PCA spots
`area, mm2
`resulting in blueing
`lesions1
`
`ODX3 (1:500)
`ODX3 (1:500)
`ODX3 (1:500)
`A5-1 (1 :100)2
`A5-1 (1:100)
`A5-1 (1:100)
`A5-1 (1:100)
`
`150
`15
`1.5
`150
`15
`1.5
`0.15
`
`128
`32
`32
`296
`32
`32
`32
`
`324
`289
`144
`280
`227
`113
`0
`
`100
`100
`25
`100
`100
`53
`0
`
`1 Percentages given are approximations.
`2 These data are taken for comparison from R ichter [1970].
`For antiserum ODX3, the amount of antidextran injected per skin site, was 0.26 fig.
`
`of the 60 titrations summarized in table II. The latter table also shows that,
`with serum dilutions which produce PCA lesions of about 10 mm diameter
`or more, 100% of sensitized sites resulted in blueing lesions. With weaker
`serum dilutions, producing PCA lesions of average diameter 3 mm, only
`about 50% of sensitized sites resulted in lesions.
`PCA titrations of antisera with known content of precipitating antidex­
`tran are shown in table III. The amount of antidextran injected per skin site
`in the various dilution steps, comprising the ‘useful dose range’ for PCA,
`averaged from 0.05 to 0.70 fig. The smallest amount of antidextran detected
`was 0.02 fig (antiserum KP8).
`Figure 1 demonstrates that an approximately linear relationship was
`obtained between the diameter of PCA lesions and the logarithm of anti­
`dextran concentration.
`In tables V-VII, results from six series of PCA experiments in guinea
`pigs are presented, showing the activity of various IO and dextran fractions
`as elicitors of cutaneous anaphylaxis in passively sensitized animals. For
`sensitization, three rabbit antidextran antisera (HDX1, ODX3, and KPX)
`were injected i.d. in two dilutions each. The corresponding amounts of
`antidextran injected per site were 0.26-0.36 fig for the lower, and 0.87-1.20
`fig for the higher dose. The potential elicitors were all given i.v. at a dose
`level of 150 fig/kg.
`
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`258
`
`R ichter
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`Relative antidextran concentration
`
`Fig. 1. Relationship between log concentration of sensitizing antidextran and diameter
`(mm) of PCA lesions on challenge with 150 //g/kg of dextran Mw 71.000. Each point in
`the graph is the average from 84 to 420 individual PCA lesions.
`
`Dextran Mw 71,000 was included as reference polyvalent elicitor and
`provoked large PCA lesions in 100% of sites sensitized with either of the
`three antisera.
`As all potential elicitors were given at the same dose of 150 /¿g/kg, this
`meant a larger dose in moles for the small molecular oligosaccharides as
`compared to the large molecular reference dextran with Mw 71,000-Mn
`57,200. The relative number of moles of the IO and dextran fractions are
`given at the bottom of table V.
`With the large dose of 150 /¿g/kg of the polyvalent dextran Mw 71,000,
`the size of PCA lesions is determined mainly by the amount of sensitizing
`antibody, as shown in tables II and III. It can be seen from the data of
`tables V-VII that the amount of antidextran injected for sensitization, cor­
`responds to the medium to high useful dose range for PCA, resulting in
`lesions with average areas of 289-441 mm2 (17-21 mm diameter) on chal­
`lenge with dextran Mw 71,000.
`Among the different oligosaccharides, it is apparent, that isomaltohexaose
`and IO of smaller molecular size were devoid of anaphylactogenic effect on
`challenge in animals sensitized with either of the three antisera injected at
`both the lower and the higher antidextran dose level.
`In contrast, isomaltodecaose proved non-anaphylactogenic in animals
`sensitized with the smaller dose of antidextran, but was clearly anaphylacto-
`
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`Molecular Size of Dextran Eliciting PCA in Guinea Pigs
`
`259
`
`Table V. Molecular size and capability of 10 and dextran B 512 fractions to elicit PCA on
`i.v. challenge in guinea pigs, sensitized with varying amounts of rabbit antidextran anti­
`serum HDX1. A fixed challenging dose of 150 //g/kg of 10 or dextran fractions was given
`
`Sensitizing Carbohydrate Mw
`antiserum
`tested
`dilution
`HDX1
`
`Mn
`
`Relative PCA
`Number PCA lesions
`reactivity1
`of spots area) coiour
`mm2 intensity dextran Mw
`0,1,2,3,4 71,000 = 100
`
`1:1,000
`1:1,000
`1:1,000
`1:1,000
`1:1,000
`1:1,000
`1:300
`1:300
`1:300
`1:300
`
`828 24
`isomaltopentaose 828
`1,152 24
`isomaltoheptaose 1,152
`1,638 24
`isomaltodecaose 1,638
`2,670 48
`dextran fraction 3,100
`6,100 24
`dextran fraction 10,500
`dextran fraction 71,000 57,200 48
`isomaltohexaose
`990
`990 24
`1,638 24
`isomaltodecaose 1,638
`dextran fraction 3,100
`2,670 24
`dextran fraction 71,000 57,200 24
`
`9
`25
`49
`225
`289
`361
`36
`289
`256
`441
`
`0.3
`0.8
`1.2
`2.5
`2.8
`2.9
`0.8
`2.5
`2.9
`2.8
`
`2
`7
`14
`62
`80
`100
`8
`66
`58
`100
`
`1 Calculated from the area of PCA lesions.
`Antiserum HDX1 contained 3.6 mg/ml of precipitating antidextran. When injected
`i.c. (0.1 ml) in dilutions of 1:1,000 and 1:300, this corresponds to an antidextran dose of
`0.36 and 1.20 /¿g/site.
`If the challenging dose of various elicitors shall be expressed on a molar base, Mn has
`to be used instead of M,v for calculations with dextran fractions. Compared on a weight/
`weight base, the relative molarities of reference dextran Mw 71,000-Mn 57,200 and that
`of the other elicitors are as follows (Mn in brackets) : dextran (57,200) = 1, dextran (33,900)
`= 1.7, dextran (6,100)=9.4, dextran (2,670)=21, dextran (1,135) = 50; isomaltose oligo­
`saccharides: dodecaose ( 1,962)=29, decaose (1,638) = 35, octaose (1,314)=44, heptaose
`(1,152) = 50, hexaosc (990) = 58, pentaose (828)=69, triose (504)= 113, D-glucose (180) =
`318.
`
`genic at the larger antidextran dose level. The same pattern could be observ­
`ed with all three rabbit antidextran antisera: HDX1, ODX3, and KPX.
`All oligosaccharides or dextran fractions of a molecular size exceeding
`that of isomaltodecaose gave strong PCA lesions, with areas larger than
`100 mm2 on challenge in guinea pigs sensitized either with the smaller or
`larger dose of antidextran. An exception was antiserum ODX3 which, when
`used for sensitization at the lower dose of 0.26 ¿zg/site, required a dextran
`fraction of a minimal size of Mw 10,500-Mn 6,100 for elicitation of a strong
`PCA response.
`
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`260
`
`R ichter
`
`Table VI. Molecular size and capability of 10 and dextran B 512 fractions to elicit PCAon
`i.v. challenge in guinea pigs, sensitized with varying amounts of rabbit antidextran anti­
`serum ODX3. A fixed challenging dose of 150 tig/kg of 10 or dextran fractions was given
`
`Carbohydrate
`tested
`
`M ,
`
`Mn
`
`Sensitizing
`antiserum
`ODX3,
`dilution
`
`Number
`of spots
`
`PCA lesions
`area, colour
`mm2 intensity
`0,1,2,3,4
`
`Relative PCA
`reactivity,1
`dextran Mw
`71,000=100
`
`1:500
`1:500
`1:500
`1:500
`1:500
`1:500
`1:500
`1:500
`1:500
`1:150
`1:150
`1:150
`1:150
`1:150
`1:150
`1:150
`1:150
`
`180 32
`180
`D-glucose
`504 24
`504
`isomaltotriose
`990 32
`990
`isomaltohexaose
`1,314 32
`1,314
`isomaltooctaose
`1,135 32
`1,430
`dextran fraction
`2,670 32
`3,100
`dextran fraction
`6,100 32
`10,500
`dextran fraction
`40,200 33,900 32
`dextran fraction
`71,000
`57,200 64
`dextran fraction
`180
`180 24
`D-glucose
`666 24
`666
`isomaltotetraose
`990
`990 24
`isomaltohexaose
`1,430
`1,135 24
`dextran fraction
`1,638
`1,638 48
`isomaltodecaose
`1,962 48
`isomaltododecaose 1,962
`dextran fraction
`3,100
`2,670 48
`71,000
`57,200 48
`dextran fraction
`
`0
`0
`0
`9
`36
`49
`289
`361
`324
`16
`9
`0
`256
`225
`256
`256
`324
`
`0
`0
`0
`0.4
`0.5
`0.8
`2.8
`3.0
`3.0
`0.4
`0.3
`0
`2.8
`2.3
`2.7
`2.1
`2.3
`
`0
`0
`0
`3
`11
`15
`89
`111
`100
`5
`3
`0
`79
`79
`79
`79
`100
`
`1 Calculated from the area of PCA lesions.
`Antiserum ODX3 contained 1.3 mg of precipitating antidextran per ml. When in­
`jected into the skin (0.1 ml) in dilutions of 1:500 or 1:150, this corresponds to an anti­
`dextran dose of 0.26 and 0.87 /rg/site.
`
`In table VIII, some hapten inhibition experiments of PCA are sum­
`marized, using isomaltohexaose as inhibitor. Tt is evident that isomalto-
`hexaose per se is incapable of eliciting PCA, whereas isomaltodecaose elicits
`strong PCA reactions with lesion areas from 225-289 mm2. When a mixture
`of both oligosaccharides was used for challenge, inhibition of PCA was
`obtained in animals sensitized with either of the three antisera KPX, ODX3,
`and HDX1. In animals sensitized with ODX3, 100% inhibition of PCA was
`seen on challenge with a mixture of 1.5 mg/kg of hexaose + 0.15 mg/kg of
`decaose. Using the same mixture and dosage of the two oligosaccharides for
`challenge, complete inhibition was seen in animals sensitized with antiserum
`KPX. When the dose of the admixed isomaltoheaose was reduced to
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`Molecular Size of Dextran Eliciting PCA in Guinea Pigs
`
`261
`
`Table VII. Molecular size and capability of IO and dextran B 512 fractions to elicit PCA
`on i.v. challenge in guinea pigs, sensitized with varying amounts of rabbit antidextran
`antiserum KPX. A fixed challenging dose of 150 /rg/kg of 10 or dextran fractions was given
`
`Sensitizing
`antiserum
`KPX,
`dilution
`
`1 300
`1 300
`1 300
`1 300
`1 300
`1 100
`1 100
`1 100
`1 100
`
`Carbohydrate
`tested
`
`M ,
`
`Mn
`
`Number
`of spots
`
`PCA lesions
`area, colour
`mm2 intensity
`0,1,2,3,4
`
`Relative PCA
`reactivity,1
`dextran Mw
`71,000=100
`
`isomaltohexaose
`990 24
`990
`1,638 24
`1,638
`isomaltodecaose
`3,100
`2,670 24
`dextran fraction
`6,100 24
`dextran fraction 10,500
`dextran fraction 71,000 57,200 24
`isomaltohexaose
`990
`990 24
`isomaltodecaose
`1,638
`1,638 24
`dextran fraction
`3,100
`2,670 24
`dextran fraction 71,000 57,200 24
`
`0
`0
`121
`121
`289
`0
`225
`196
`361
`
`0
`0
`1.7
`1.2
`2.6
`0
`2.5
`2.2
`2.5
`
`0
`0
`42
`42
`100
`0
`62
`54
`100
`
`1 Calculated from the area of PCA lesions.
`The content of precipitating antidextran in antiserum KPX was 0.9 mg/ml. When
`injected i.d. in dilutions of 1:300 and 1:100 (0.1 ml), this corresponds to an antidextran
`dose of 0.30 and 0.9 /rg/site.
`
`0.19 mg/kg, there still occurred an approximately 50-percent reduction in
`size of PCA lesions. The molar ratio between hexaose and decaose in the
`latter experiment was 2.1:1.
`In contrast, the PCA-eliciting effect of isomaltodecaose in animals
`sensitized with HDX1 was more difficult to inhibit and a much larger molar
`excess of the isomaltohexaose was required. A mixture with a molar ratio of
`8.3:1 reduced the size of PCA lesions only insignificantly. In a second
`experiment, a significant reduction of PCA area of about 50% was attained
`with a molar ratio of 83:1.
`
`Discussion
`
`Simplicity and high sensitivity make PCA a most valuable tool for in vivo
`studies of the mechanisms of immediate-type allergic reactions. This proce­
`dure has been named, introduced and developed by O vary [1952, 1958,
`1964] and has been extensively employed by O vary and coworkers [Bena-
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`262
`
`R ichter
`
`Table VIII. Inhibition of the anaphylactogenic effect of isomaltodecaose by isomaltohexaose
`admixture on challenge in guinea pigs sensitized i.d. with rabbit antidextran antisera
`
`Sensitizing
`antiserum
`code
`dilution
`
`Oligosaccharide used
`for challenge (mg/kg)
`
`Number
`of spots
`
`PCA lesions1
`area, colour
`mm2
`intensity
`0,1,2,3,4
`
`Relative PCA
`reactivity,2
`isomalto­
`decaose = 100
`
`KPX
`KPX
`KPX
`
`KPX
`
`1:100
`1:100
`1:100
`
`1:100
`
`KPX
`
`1:100
`
`KPX
`
`1:100
`
`ODX3
`ODX3
`ODX3
`
`HDX1
`HDX1
`HDX1
`
`1:150
`1:150
`1:150
`
`1:300
`1:300
`1:300
`
`HDX1
`
`1:300
`
`(0.15)
`isomaltohexaose
`isomaltodecaose
`(0.15)
`isomaltohexaose
`(1.5)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(0.75)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(0.38)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(0.19)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(0.15)
`isomaltodecaose
`(0.15)
`isomaltohexaose
`(1.5)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(0.15)
`isomaltodecaose
`(0.15)
`isomaltohexaose
`(0.75)
`+ isomaltodecaose (0.15)
`isomaltohexaose
`(7.5)
`+ isomaltodecaose (0.15)
`
`24
`48
`
`48
`
`48
`
`24
`
`48
`24
`48
`
`32
`24
`24
`
`48
`
`48
`
`0
`225
`
`9
`
`25
`
`0
`
`100
`0
`225
`
`0
`36
`289
`
`225
`
`121
`
`0
`1.9
`
`0.4
`
`0.5
`
`0
`
`1.2
`0
`2.3
`
`0
`0.8
`2.5
`
`2.2
`
`1.5
`
`0
`100
`
`4
`
`11
`
`0
`
`44
`0
`100
`
`0
`12
`100
`
`78
`
`42
`
`1 Average from all sites injected.
`2 Calculated from area of PCA lesions.
`At the dilutions used above, the amount of precipitating antidextran injected per site
`Gug) was as follows: KPX=0.90, ODX3=0.87, HDX1 = 1.20.
`
`cerraf et at., 1963; Ovary et al., 1963] in elucidating the homocytotropic
`properties of guinea pig antibodies. Ovary and Karush [1960] also first
`obtained hapten inhibition of PCA in guinea pigs. In humans, specific
`inhibition of wheal and erythema responses with univalent hapten has been
`reported by Farah et al. [I960].
`With the strong rabbit antidextran antisera used in this study, and
`employing a fixed large dose of antigen, the smallest amount of sensitizing
`antidextran, still producing a positive reaction in about 50% of sites in­
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`Molecular Size of Dextran Eliciting PCA in Guinea Pigs
`
`263
`
`jected, was found to be 0.02-0.09 fig. This corresponds in order of magnitude
`to the figures given by O vary and Briot [1951] for rabbit anti-ovalbumin.
`When using a large dose of multivalent antigen (dextran Mw 71,000) for
`challenge, an approximately linear relationship was obtained between dia­
`meter of PCA lesions and log dose of sensitizing rabbit antidextran. Such a
`relationship is also mentioned by Brocklehurst [1967] in a description and
`discussion of the PCA procedure.
`The aim of this study was to elucidate the smallest molecular size species
`of dextran and of purified oligosaccharides of the isomaltose series required
`to elicit PCA in sensitized guinea pigs. To facilitate experimentation and
`interpretation of results, it was decided to use a large fixed amount of sen­
`sitizing antidextran, and a large dose of the potential elicitor for challenge.
`The antidextran amount was such that it produced large PCA lesions in
`100% of animals, upon challenge with 150 fig/kg of the multivalent reference
`dextran fraction of Mw 71,000. The latter dextran dose was about 100 times
`larger than that required to give PCA lesions in 25-50% of sites sensitized
`with a large antidextran dose. It has been shown earlier, that an excessive
`dose of challenging dextran of Mw 71,000 (150,000 fig/kg) does not diminish
`the size of PCA lesions in sensitized guinea pigs [R ichter, 1970], Thus, when
`no PCA lesions are obtained with a potential elicitor under the conditions
`chosen in this study, the activity of that elicitor is at least 100 times weaker
`than that of the multivalent reference dextran.
`On testing IO of increasing molecular size for their ability to produce
`PCA lesions, negative results were always obtained up to the isomalto-
`hexaose. Isomaltodecaose, on the other hand, gave strong PCA lesions with
`all three antisera at the higher, but negative results at the lower antidextran
`concentration. From the extensive studies of R abat and coworkers [R abat,
`1954, 1956, 1960; M age, 1963; M age and R abat, 1963], it is known, that
`the binding site of rabbit and human antidextrans is complementary to a
`sequence of 3-6 glucose residues on the dextran molecule.
`Further evidence for this size of the antibody-binding site is provided
`by the findings of A rakatsu et al. [1966]. These workers found that iso-
`maltotrionic acid, when covalently coupled to a protein carrier, upon im­
`munization of rabbits, elicits formation of antibodies, cross-reacting with
`large molecular size dextran. Isomaltonic acid, similarly coupled to a carrier,
`produced antibodies specific for isomaltose, but no cross-reactivity with
`dextran was observed.
`The results obtained with isomaltodecaose in this study are in accordance
`with the above findings, regarding the size of the antidextran-binding site.
`
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`264
`
`R ichter
`
`In the presence of a high local concentration of cell-fixed antidextrans,
`isomaltodecaose is capable of bridging two binding sites of two adjacent
`antidextran molecules, thus triggering the subsequent steps which finally
`lead to release of vasoactive mediators. Regarding mechanisms of elicitation
`of immediate-type reactions see e.g. O vary and K arush [1960], Stan-
`w o rth [1963, 1970], Levine [1965], L evine and F ellner [1965], O vary
`[1965], de W eck [1968], de W eck and Schneider [1969], Ishizaka and
`Ishizaka [1969], I shizaka et al. [1964],
`As isomaltohexaose never produced PCA lesions, and also has been
`shown to inhibit precipitation of antidextran by large molecular size dextran
`[see e.g. M age, 1963; R ichter, 1971a], it was thought interesting to study
`whether the PCA lesions induced by isomaltodecaose could be inhibited by
`isomaltohexaose, admixed to the challenging solution. This proved to be
`the case, and the ease with which the hexaose inhibited PCA reactivity of
`the decaose in some antisera, indicated that the binding forces between
`decaose and antidextran were relatively similar to those between hexaose
`and antidextran. However, the important difference between the two oligo­
`saccharides was the ability of the decaose to bridge two binding sites between
`two adjacent antidextran molecules, and the lack of this ability in the
`hexaose. A molar ratio of 2:1 between hexaose and decaose was sufficient
`to produce reduction of average PCA area with 50%, using antidextran
`antiserum KPX. With the same antiserum, higher hexaose concentrations
`when admixed to isomaltodecaose, completely inhibited PCA.
`On sensitization with the antiserum HDX1, a much higher dose of hexa­
`ose was required to inhibit PCA lesions provoked by the decaose. With a
`molar ratio of 83:1, the average area of lesions was reduced with 50%. It is
`probable that the higher dose of hexaose required for inhibition of PCA in
`this case, reflects a higher affinity of antidextran antibody populations in
`antiserum HDX1, which was raised by immunizing rabbits with a hemo-
`cyanin-dextran conjugate.
`From the fact that isomaltohexaose and isomaltoheptaose have been
`shown to inhibit precipitation of antidextran by large molecular size dextran
`in vitro [K abat, 1962; R ichter 1971a] and from the demonstrated PCA
`reactivity of the isomaltodecaose in this study, it is evident that the transi­
`tion from non-elicitor to elicitor molecules is rather abrupt. This may imply
`that the majority of subpopulations of antidextran antibodies have binding
`sites complementary to at least 3-6 residues, and that the decaose is the
`smallest molecule capable of bridging a critical number of antidextran
`molecules, sufficient to trigger the subsequent disturbances in the membrane,
`
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