.an Utympus BH2 microscooë equrpped with a Fairchild CCD camelà connected to an AT computer with an. Jvtatrgx_. MVP/AT imagè pi*"*i* board. Software for ...
Microecology and Therapy
Milroökologie
unA
-
theràpie
4e9
(lee0i
20, 4es t_o_I.
rssN 0720-0536
Edited by
Instinrt fiir Mikroökologie D 6348 Herborn-Dill
Germany
CHARACTERIZATJOI! THE INTERINDIVIDUAL DIVERSITY OF THE SERU}T--A-NTIBÀö -OT TiiIAT ANTIBODY RESPONSE AGAINST THEJNDTGENOUí iNTE§TINAL MICROFLORA OF HEALTHY VOLUNh-ÈÈNS''^'HERMA Z. APPERLOO--RE-NKEMA, THEA MTcHAEL H.F. wy:FrNSoN, BÀhï c.- G. JAGT, rvreiÈÈ,^' andDIRK VANDER\ryAAIJ
INTRODUCTION The composition of the indisenous mrcïoflora of the intestinal Eact iíoar:tlv .
tne characterisdc
óíö;.,, *9ry.*.0 ot the host. !v In mice e.g. a cross_rèactiv_ ity was shown between the foeial colonic mucosa and Bacteror.d^ ;;i; colonizin-g adult mice (t). ih.-HLï pattern of the host may aÍso influence the composition of thË ;iiliil;^;; was- shown in a study o, t*ini-*iit, Crohn's.disease (2). e p*ticriai Hf_ï pattern is sometimes correlated with a specific disease as well a, *itt u teristic composition of the, fàË;i;"f,*url c.rgtloÍa. In humans a relationshio of this kind has been shown-in anili,rólr,iË spondylitis, which occurs more óften if; patienrs -canying HLA-827, *iih ;ilrL taneously Klebsiella in their faeces 13,4). Analogous to studies on HLA_ antlgens- (5), we investigated whether we could characterize hurÀan inaiviàuàli with respect to the serum antibodies ài_ ypa ruuilst the indigenou, ..,i.iàno* 9r th9 lntestinal tract of other volunteers. Our hypothesis is that a person with low serum antibody titres àgainst his/her gwll uoJa, may have higher serum anri_ Dooy tttres against bacteria of the rndlgenous faecal microflora of unre_
lated persons.
T!is. study may provide a new wav insight in the interaction bJ_ tween host individuals and human mi_ ot^ galning
croflora. In autoimmune diseases in addition to the genetic prop".ti"r,"tt rndlgenous microflora compösition may" play a role.
MATERIALS AND METHODS Eight healthy volunteers, five íale and three t'en11]e, participated in the
srudy by providing one faècd and one otood sample. Diluted serum (l:16)
of gaclr volunreer was absoruJo-íitr, herlhis own faeces, ufto *Íi.f, ,"*rn antibodies of IgA,IgG and I;Mlr;À;..
were measured against the -bacterià in tne washed faeces of the other seven volunteers by indirect irn-unonroi# cence. As a positive control we assessed m^e presence of antibodies in the serum of e_ach volunteer against his-;;;nri. crqflgra without abJorption of *iiUao terial antibodies. We pë.fo.rnà iralr., immunofluorescence irtro *itf, uUroAË serum and faeces of the same ,roluntee, as an absorption control anO *àihà taeces with pBS instead of serum as i negative PBS control. slides were read by ^.. l:gooscopic an rmage processing system consistini or .an Utympus BH2 microscooë equrpped with a Fairchild CCD camelà connected to an AT computer with an Jvtatrgx_
MVP/AT imagè
pi*"*i* ;a;;:
board. Software for reaiinf
uating data was developed in-orn-laË_ tory (6). The softwaré combines mea_
49ji
Table 1A: his
t
_______{ fmes ofA; volunteer
I
a
§;
,()É\
2l ..;
,Isit 3
.;
I I
t9
rï
CU
LT o
?
r
?*t, ltJ v
wI
*/, t-
]r >ï
a
/q
J
,
A
_*
B
235
C
5
D
(o LL
-1.0 0.0 r.0 2.0 3.0 4.0 5.0 6.0
7.0
Facton t--> 10
E
25
F
r25
pm
Figure 1: Faecal bacteria of volunteer C are classified in five clusters based on their
G
morphological properties.
H
suring of micromorphology of faecal bacteria with scoring of IIF of each bacterium.
For each combination of faeces of a volunteer and autologous-flora absorbed serum of another volunteer 500 - 1000 bacteria were analysed both with regard to their individual morphology and to the occurrences of fluorescence due to
positive indirect immunofluorescence. The bacteria analysed belonged to the anaerobic indigenous microflora of the respective volunteers. Based on micromorphological properties the bacteria were classified in clusters. Cluster 1 contains coccoid and small rods, cluster 2 contains small cocci, cluster 3 large
cocci, cluster 4 long slim or irregular rods and strings of cocci and finally cluster 5 contains intermediate rods (Figure 1). Within theseglusters the median of fluorescence ,íntensity of the bacteria was measured. In this way it was possible to assess humoral immunological reactivity of volunteers against the indigenous microflora of others.
496
23,J 5
RESULTS
The results are shown in tail 18 and lC. Concerning clusur 2, there was no difference in il quency in which IgA, IgG adI! tibodies were directed againre However, IgG antibodies often directed against bacteria to cluster 3 than IgA and IgM
ies; the opposite was found 4. IgM antibodies were Íx)ÍG sured against cluster 5 than antibodies.
fu
DISCUSSION AND SION Our method provides the to characterize human indiv cerning their humoral activl intestinal bacteria in a similr commonly used in tissue Like reactivity to unrelarcd gens, there appears to be m vidual difference in lated (human) indigenous r i.e. with respect to the senm directed against the indi
*The numbers in absorbed serum-
'
faeces
of volunteer
A B C
D E
2,31 5
F
2,3§ 5
G
H
4
t:
"!
.ï
Tabte 1A: Circulating IgA Antibodies Present in Serum of a Volunteer after Absorption with his Own Faeà Microflora Reactive against Components of the Faecal Flora of Unrelated Healthy Volunteers
positive
absorbed serum of volunteer
frces
ofAB
CDEF
conEol
volunteer
A
_:1.
B
2,3,5
C
5
1,2,3
2,4,5
4,5 2,3,5
2,4,5
4,5
4
4
3A 2,3,4
4
D
2,4,5 _ -4 -
4
5
E
2,3,4
2,5
2,4
5
2,34
F
1,2,5
G
2,3,4
4
H
:
2,4
2,4,5 1,2,3
2,4,5
2,3,5
3'4,5 1,2,3 4,5 1,2,3 4,5 2,3,4 5
2,3,4 5
2,3
4,5 *The numbers in the tables indicate clusters of faecal bacteria against which antibodies are present in the absorbed serum,
Table 18: Circulating IgG Antibodies positive
absorbed serum of volunteer
faeces
CDEF
of
control
volunteer
A B
2,3,4
3,s
3,5
2,3,4
2,3,4
1
2,3
5
2,3,4
2,3,4
:
5
C
)?
:
D
2
2,34
2,5
5
2
5
E
1,2,4
2,3,4
t,2,3
5
4,5 2,4,5
2,3,5
2,3,4
J
J
2,4,5
5
F
2,4,5
5
5
G H
4 _
2,3,4
5
2,3,4
2,3,4
5
5
-
2,3,4 5 5 3,5 2,3,4 5 2,3 r,2,3 4,5 2,3,4 2,5 L,2,3 4,5 5 2,5 2,3,5 1,2,3 4,5 2,3,4 5 5 2,4,5 5 2,3,4 2,3,4 55
497
lC:
Table
of Groningen,
Circulating IgM Antibodies
EZ Groningen,TTE
absorbed serum of volunteer
faeces
cd
CDEFG
of A B
2,3,4
c D E
2,3,5
F
2,5 5
G
544 4,5 2,5
4
5
4
2,5
2,3A L\l 5
2,3,5
44-
2,5 2,3A s15
rz?
4,5
2,4
4,s
3,5
2,3,5
3
_
assessed. -- -óiif"t"nces
existed interindividually differences with inraindividual Uut-Jsó resDect to the isotypes were tound' rt appeared that IgG antibodies were more óïírí àir".t"a alainst bacteria belonging ió-ótuit"t 3 thai IgA and IgM antibodies. This might indicate that the locat
ií*unity
fínctions in-adequately in
confinin§ the bacteria of this cluster to the diee;tive tract; i.e. the bacteria may 6" uUÉ to Eanslocate to the mesenteric iy-pttnoA"s and/or the spleen to induce
498
G r o ni
3,4,I
ng e n,"Ó o ster sing-el 59, nï e n, T he N e therlands'
3 EZ Groningen, Tle
Nfr
Bart C. Meiier, LaboratorYfu
MicrobiolàgY, State q"!Y G r o ni ng e n,"Ó o ste r s ingel 59, G r o ni n§
e
n, T he N ethe rlands'
Dirk van der Waaii,
M e dical M icrobiolo gY,
Stac
Foo, M.C., and cross-reaction testine and a tochthonous
Immun.9, I Van de Merwc" J.H., and resident faeal by genetic host. Imp ease? Antonic
45
of Groningen, Qolg
1
2.
L4r
utchaël H.F. Wilkinson, L for Medical MiuobiologY, "versity 97
l.
12"5 45
MicrobiotàgY, State Unívr Gr o ni
LITERATURE
123
antiMies against these bacflora of the intestinal tract of other vol- IeG - Finally not all clusters qPPq unteers. -immunogenicitY' I equal oo*ti i, uaaition the present study reveals 'no antiUo«iies were found agaiÉ other interindividual diferences: occain cluster 1. This iionally, a person with an indigenous ria classified öf low immunogenicity e1- relatively low immulogeniorY -hitiG -ictofÍotu belonging to this clusm' a hieh level of anitbodies in the bacteria uuioiuta sËrum against the faecal baci"iiu or other volinteers. On the other H erma Z. APPerloo-Rer*enn, irïrà u p"*n with almost no antibodies tom for Mààtcal Microbiolal i, r,itótouued serum against the faecal tJitierstty of Groningen, 0-6 DacÏaecal bacteria of others, may have gzti oZ Groningen, Tl* teria against which the other persons ig, have àanY antibodies. However, no lands. correlations could be inea G. Jagt, LaboratoryÍY
rig"ifi*"1
21, 1990,I*iden,
45
rzr 2,4 45
-
2,4 2,4
2,4
t
