Immunologic Studies in Pernicious Anemia - Blood

Immunologic Studies in Pernicious Anemia

By Ciu�ua TAI AND JAMES E. MCGUIGAN

M ANY INVESTIGATORS have described antibodies to intrinsic factor

( IF ) in sera from patients with pernicious anemia.’4 These antibodies2

have been classified as “blocking” and “binding” antibodies. Antibodies to IF

designated as blocking antibodies are those which combine with IF inhibiting

subsequent complex formation between vitamin B12 and intrinsic factor. Bind-

ing antibodies are those which bind intrinsic factor molecules before or follow-

ing the complexing of intrinsic factor with vitamin B,2 but do not prevent

formation of intrinsic factor-vitamin B12 complexes. In addition to antibodies

to intrinsic factor there are in the sera of approximately 90 per cent of patients

with pernicious anemia antibodies to parietal cell cytoplasmic constituents57

which have been detected by complement fixation5’6 and immunofluorescent

technics.#{176}’7

Detection of intrinsic factor and parietal cell antibodies, in addition to

certain clinical and morphologic findings in pernicious anemia, has raised the

question whether pernicious anemia is an autoimmune disease, i.e., a disease

in which immunologic phenomena play an integral role in either the initiation

or perpetuation ( or both ) of pathologic events in the disease process. To date

there is no direct evidence to support the speculation that antibodies to in-

trinsic factor play an etiologic role in the pathogenesis of the gastric atrophy

which characterizes classical adult Addisonian pernicious anemia. In 30 per

cent to 50 per cent of patients with pernicious anemia, antibodies to intrinsic

factor cannot be demonstrated with currently available methods. There has

been no correlation established between the absence, presence, or levels of

antibodies to intrinsic factor and the clinical course of patients with pernicious

anemia.8’#{176} Furthermore to date there is no convincing evidence that circulat-

ing antibodies damage normal tissue components in the absence of lymphocyte

10,11

In studies of experimental autoimmune disease passive transfer of the im-

munologic disease has been accomplished by transfer of sensitized lympho-

This work was supported in part by Research Grant 1RO1 AM 10837 froni the National

Institutes of Health (Dr. McGisigan), by Grant T-394 frcnn The American Cancer Society, by

Grant-In-Aid 66 679 from The American Heart Association, and by Research CareerDevelopment Award 7-K3-AI-19,499 from The National Institutes of health (Dr.

McGuigan).

First submitted August 21, 1968; accepted for publication March 10, 1959.

CHIAKI TA!, M.D., PH.D. : Research Instructor, Departntent of Medicine, WashingtonUniversity School of Medicine, St. Louis, Mo.; present address: Department of Surgery,

Okayama Medical Sc/tool, Okayarna, Japan. JA�n�s E. MCGUIGAN, M.D. : Assistant Professor

of Methcine, Washington University School of Medicine, St. Louis, Mo.; present address:

Divison of Gastroenterology, De-partm�nt of Medicine, University of Florida College ofMedicine, Gainesville, Fla.

Address reprint requests to: Dr. James E. McGuigan, Division of Gastroenterology, Depart-ment of Medicine, University of Florida, College of Medicine, Gainesville, Florida.

BLOOD, VOL. 34, No. 1 (Juix), 1969 63

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64 TA! AND MCGUIGAN

cytes’2 but not by transfer of antibodies; delayed type hypersensitivity is

believed to play the crucial role in the induction of a variety of autoimmune

states.

We have examined the lymphocyte transformation1317 characteristics of

patients with pernicious anemia in order to identify a potential role for cellular

immunity in pernicious anemia. When small lymphocytes in culture are ex-

posed to an antigen to which they are immunologically sensitized or when

they are exposed to a nonspecific mitogen such as phytohemagglutinin (PHA)17

they undergo morphologic and functional changes designated as lymphocyte

transformation. The lymphocytes increase in size, the nuclei become more

esinophilic, the cytoplasm more basophilic. Prominent nucleoli appear. Vac-

uoles are seen in the cytoplasm adjacent to the nucleus. In association with

these morphologic changes there is increased synthesis of RNA and protein

and increased uptake of tritiated thymidine into DNA. In experimental

studies lymphocyte transformation demonstrates a high degree of correlation

with delayed ( cell mediated ) sensitivity.18

MATERIALS AND METHODS

Patients

A group of twenty-nine patients with classic Addisonian pernicious anemia with the

diagnosis established by customary clinical and laboratory criteria were evaluated in thisstudy. The patients included 7 males and 22 females, from 39 to 88 years in age. All pa-tients had a clinical history of pernicious anemia exceeding three years in duration. All

patients were being treated with monthly intramuscular injections of vitamin B12. Eighteen

hospitalized patients of similar age distribution to the patients with pernicious anemia were

utilized as control subjects.

Antibody Studies

Determination of Autoantibodies in the Sera of Patients with Pernicious Anemia: 1 ) Block-ing antibodies to intrinsic factor. The presence of blocking antibodies to intrinsic factor was

determined by use of the charcoal-assay technic.18 Blocking antibodies to intrinsic factor

were considered to be present when 0.1 ml of the patient’s serum reduced by more than 50

per cent the binding of 2 ng of �57Co]-cyanocobalamin by intrinsic factor contained in

human gastric juice. 2) Binding antibodies to intrinsic factor. Binding antibodies to intrinsic

factor were determined utlizing the radioimmunodiffusion technic as modified from

Samloff et al.4 3) Antibodies to parietal cell antigen. The microsomal parietal cell antigen

was purified from human gastric mucosa by use of the ficin method as described by Baur

et al.1#{176}The presence of antibodies to the microsomal parietal cell antigen was determined

by using complement fixation technics using fresh sheep red blood cells.20

Lymphocyte Culture and Transformation Studies

Antigen preparations used in lymphocyte cultures. 1. Human gastric juice (homologous)

was collected following subcutaneous administration of 50 mg of Histalog ( Lilly ) by

aspiration with a gastric tube after an overnight fast. Gastric juice was collected by manual

aspiration and immediately placed into an ice bath. To inactivate pepsin activity the gastric

juice was adjusted promptly to pH 10 by addition of 1 M sodium hydroxide. The gastric

juice was maintained at pH 10 for 30 minutes on ice and then adjusted to pH 7 by addition

of 1 M hydrochloric acid. Pooled neutralized gastric juice was then extensively dialyzed

for 48 hours at 4 C. against 0.15 NaCl-0.01 M potassium phosphate, p11 7.4 (phosphate

buffered saline). Intrinsic factor content was measured by the charcoal assay technic18

and the protein content was estimated by optical density determinations using a Beckman




IMMUNOLOGIC STUDIES IN PERNICIOUS ANEMIA 65

DU-2 Spectrophotometer at 280 m�z ( standarized with bovine serum albumin:

E � �m. 6). The IF binding capacity of the gastric juice preparation was 61 ng. vitamin

B12 per ml. 2. Highly (but not completely) purified intrinsic factor for use as antigen wasprepared from human gastric juice according to the method of Chosy and Schilling2’ using

a series of column fractionation procedures. The IF binding capacity of these preparations

were 300 to 500 ng vitamin B12 per ml. 3. Normal human gastric mucosa was prepared

from surgically resected human stomach kept at -20 C. until use. The mucosa following

separation from the underlying submucosa was minced in 0.15 M NaC1 and then rapidly

frozen and thawed ten times by alternate rapid passage from a 37 C. water bath to a bath

containing dry ice and acetone. The resultant freeze-thawed homogenate was centrifugedat 700 X g. for 10 minutes. The protein content of the supernatant solution was adjusted

with 0.15 M NaCl to 250 aug. per ml. for use: the IF binding capacity was 30 ng vitamin

B12 per ml. The antigens to which the cultured lymphocytes were exposed were sterilized

by filtration through millipore filters ( N-0.4 m� ) . Following preparation of antigens they

were maintained at -20 C. until used.

Lymphocyte preparation and culture. Relatively pure preparations of lymphocytes ( 85 to

90 per cent ) were prepared from 20-40 ml. of heparinized peripheral venousblood by use of the method of Hasting and his associates.13 The lymphocytes were then

washed twice with Hank’s balanced salt solution. The lymphocytes were cultured in Eagle’s

minimum essential medium containing 20 per cent calf serum ( inactivated by heating at

56 C. for 30 minutes), 1-glutamine 30 �tg., penicillin C 200 units and potassium dihidro-

streptomycin 0.2 gig. per ml. Two ml. of the lymphocyte preparation, at a concentration of

2 X 106 cells per ml., were placed in each 15 X 1.6 cm. sterile disposable polyethylene

culture tube. Cultures were performed for 96 hr. at 37 C.

Lymphocyte transformation. Lymphocyte transformation was determined by measurement

of the uptake of [3H}-thymidine into DNA. Five hours prior to the termination of the

culture period 2 pc. of [3H]-thymidine ( specific activity 6.7 C per mM, New England

Nuclear Corporation, Boston, Mass. ) were added to each tube. At the completion of theculture period, the culture tubes were placed in an ice bucket and 2 ml. of 5 per cent

trichloracetic acid, maintained at 4 C., were added with mixing to each tube. The tubes

were then centrifuged at 4 C. at 700 X g. for 20 minutes. The supernatant solutions were

removed and discarded and the procedure was repeated. The resultant precipitates were

then washed with cold absolute methanol. Centrifugation was repeated. The precipitant wassolubilized by addition of 0.5 ml. Hyamine hydroxide followed by incubation for 20

minutes at 65 C. The solutions were transferred to 15 ml. of Bray’s solution22 and radio-

activity was measured using a Packar Tri-Carb Scintillation Spectrometer. Quantitative

estimation of lymphocyte transformation was determined by measurement of uptake of

tritiated thymidine into DNA as described by Dutton and Eady.’4 The presence andmagnitude of lymphocyte transformation were evaluated by determining the ratio of [311]-

thymidine uptake in experimental samples compared with that of control tubes to which no

antigen or PHA had been added (negative control). A ratio (E/C; E experimental,

C = negative control ) greater than 3 was interpreted as a positive response indicative of

the presence of lymphocyte transformation.

Series A. Lymphocyte cultures from 16 patients with pernicious anemia and 18 control

subjects were incubated in the presence of each of the following: 1 ) 0.1 ml. humangastric juice, 2 ) 0.1 ml. gastric mucosal homogenate, 3 ) 0.1 ml. of intrinsic factor prepara-

tion, 4) 0.1 ml. PHA-P ( 1:10).

Series B. Long-term cultures ( 7 days ) were performed with lymphocyte cultures fromfive normal individuals and lymphocyte cultures from 16 patients with pernicious anemia.

In addition to positive ( PHA-containing) and negative controls the lymphocytes were

cultured with 0.2 ml. of a concentrated gastric juice preparation with an IF. vitamin B1.,

binding capacity of 180 ng./ml. In order to assure lymphocyte viability through the

extended period of observation the culture medium was replaced with fresh culture medium

three days and five days following the initiation of the culture period.




66 TAI AND MCGUIGAN

RESULTS

Estimation of Circulating Antibodies

1. Blocking antibodies to intrinsic factor. Antibodies which blocked the

formation of [57Co]-cyanocobalamin-intrinsic factor complexes were found

in the serum of 19 to 29 of the patients with pernicious anemia and in none

of the control sera.

2. Binding antibodies to intrinsic factor. Fifteen of the 29 patients with

pernicious anemia and none of the control subjects were found to have bind-

ing antibodies to intrinsic factor in their sera.

3. Parietal cell antibodies. Of 29 patients with pernicious anemia 21 were

found to have complement fixing antibodies to the parietal cell antigen prep-

aration.

Lymphocyte Transformation

Series A. Lymphocyte cultures from 3 of 14 patients with pernicious anemia

exhibited lymphocyte transformation when cultured in the presence of purified

intrinsic factor (0.1 ml. with a vitamin B12 binding capacity of 300 ng./ml.).

Lymphocyte transformation was not observed in any of the lymphocyte cul-

tures from control subjects cultured with this intrinsic factor preparation.

Lymphocyte cultures from 2 of 29 pernicious anemia patients underwent

transformation when 0.1 ml. of homologous gastric juice ( IF vitamin B12

binding capacity 61 ng./ml. ) was included in the culture (Table 1 ) . Both of

these patients were among the three patients whose lymphocytes transformed

when cultured with the purified intrinsic factor preparation ( above ) . Lym-

Table 1.-Lymphocyte Transformation6 Studies From One Group of Nine PatientsWith Pernicious Anemia

Patient

cpmNegativeControl

cpmPHA1 : 10

cpmPHA 1 : 10

cpm NegativeControl

cpmGastric

MucosalHomogenatef

cpm GastricMucosal

Homogenate

cpm NegativeControl

cpmwith

GastricJuice

cpm withGastric Juicet

cpm NegativeControl

KI 151 51344 340#{176} 290 1.92 879 5.82*

ES 164 1849 11.3#{176} 105 0.64 144 0.88

RYI 100 2448 24.5#{176} 133 1.33 107 1.07

BL 180 36121 201#{176} 253 1.41 185 1.03

MC 283 12238 43.2#{176} 223 0.79 184 0.65

PH 268 11051 41.2* 198 0.74 156 0.58

CR 174 2295 13.2#{176} 176 1.01 187 1.07

HA 206 6312 30.6* 142 0.69 264 1.28

MOR 160 1153 7.21#{176} 142 0.89 133 0.83

0 Lymphocyte transformation was determined to be present when the ratio of uptake of

tritiated thymidine into DNA in the experimental cultures as compared with that of the

negative cultures was greater than 3.0.t The lymphocyte cultures included 0.1 ml. of the gastric mucosal homogenate prepara-

tion with an IF. binding capacity of 30 ng. vitamin B19 per ml.

t The lymphocyte cultures included 0.1 ml. of the gastric juice preparation with an IF.binding capacity of 60 ng. vitamin B12 per ml.





phocyte transformation did not occur in any of the cultures containing gastric

juice and lymphocytes from the 18 control subjects.

Lymphocytes from one of 16 patients with pernicious anemia demonstrated

lymphocyte transformation when cultured with 0.1 ml. of the gastric mucosal

homogenate preparation ( I.F. vitamin B12 binding capacity of 30 ng./ml.).

Lymphocytes from this patient also underwent transformation in the presence

of the gastric juice and purified intrinsic factor preparations ( above ) . Trans-

formation was observed by lymphocytes from one of 18 control subjects whose

lymphocytes were cultured with the gastric mucosal homogenate preparation.

Series B. Following long-term lymphocyte cultures ( 7 days ) in the presence

of 0.2 ml. of concentrated gastric juice ( I.F. binding capacity 180 ng. vitamin

B12 per ml. ) lymphocytes from 6 of 16 patients with pernicious anemia dem-

onstrated lymphocyte transformation ( Table 2 ) . These 6 patients whose

Table 2.-Transformation of Lymphocytes#{176} From Patients With Pernicious AnemiaCultured in the Presence of Concentrated Human Gastric Juicef

cpm WithConcentrated

cpm PHA 1 : 10 Gastric Juice- -� cpm withcpm Negative cpm cpm Concentrated cpm

Patient Control PHA 1 : 10 Negative Control Gastric Juice Negative Control

BAR 111 36977 333#{176} 580 5.26*

RE 418 13872 35.6#{176} 2140 5.12*

KI 1201 16131 13.4* 18737 15.6*

CO 225 5808 25.8#{176} 2199 9.77#{176}

EV 525 15558 29.6#{176} 1628 3.10#{176}

OT 511 12563 24.6#{176} 2140 4.19#{176}

* Lymphocyte transformation was determined to be present when the ratio of uptake of

tritiated thymidine into DNA in the experimental culture as compared with that of the

negative control cultures was greater than 3.0.

f Concentrated human gastric juice, 0.2 ml. (IF. binding capacity 180 ng. vitamin B12

per ml.) was included in each lymphocyte culture.

lymphocytes underwent transformation included the 3 patients whose lym-

phocytes were previously noted to transform in the presence of the purified

intrinsic factor preparation. Under these conditions lymphocyte transformation

was not observed in any of the cultures from the control subjects.

Relationship of Humoral Autoantibodies to Lymphocyte Transformation.

There was no direct or inverse relationship between the detection of anti-

parietal cell antibodies and transformation of lymphocytes from patients with

pernicious anemia. On the other hand there appeared to be an inverse rela-

tionship between lymphocyte transformation and the presence of circulating

antibodies to intrinsic factor in the serum of the patients with pernicious

anemia. Blocking antibodies to intrinsic factor were not demonstrable in the

sera of any of the pernicious anemia patients whose lymphocytes exhibited

transformation when cultured with the various gastric antigen preparations.

Binding antibodies to intrinsic factor were detected in the serum of one pa-

tient with pernicious anemia whose lymphocytes exhibited transformation and




68 TAI AND MCGUIGAN

then only following prolonged incubation with the concentrated gastric juice

preparation ( Series B, above).

DIscussIoN

Some of the best studied immunologic diseases in experimental animals

result not from tissue damage effected by circulating antibodies but are sec-

ondary to lymphocyte-mediated sensitization.12 Cell mediated sensitization,

i.e., of the delayed or tuberculin type, has not been previously reported in

patients with pernicious anemia. The technics of lymphocyte transforma-

tion1417 and macrophage immobilization� have been proposed to serve as in

vitro methods for detection of sensitization of lymphocytes. The value of these

methods, if they genuinely reflect cell mediated sensitization, as in vitro

methods to study lymphocytes from patients with diseases suspected to be

autoimmune in nature is of obvious importance. We sought to see if lympho-

cytes of patients with pernicious anemia underwent transformation when

exposed to antigens suspected to be important in the development of perni-

cious anemia.

The results of this study indicate that peripheral lymphocytes from some

patients with pernicious anemia did show evidence of delayed hypersensitivity

as judged by lymphocyte transformation. Lymphocyte transformation to hu-

man gastric juice and intrinsic factor preparations was exhibited only by those

patients whose sera did not contain circulating blocking antibodies to intrinsic

factor. In addition, of the six patients with pernicious anemia whose lympho-

cytes underwent transformation with the various gastric intrinsic factor-

containing preparations serum from only one patient contained binding anti-

bodies to intrinsic factor. On the other hand it was not possible to establish

a direct or inverse relationship between lymphocyte transformation and the

presence of antibodies to the gastric parietal cell antigen in the sera of the

patients with pernicious anemia. A satisfactory explanation for the inverse

relationship between lymphocyte transformation and intrinsic factor antibodies

cannot be offered. It is possible that if I.F. is the antigen responsible for evok-

ing lymphocyte transformation that humoral antibodies to intrinsic factor could

inhibit the expression of lymphocyte transformation by competing for IF

molecules with receptor sites on the surfaces of lymphocytes. However, the

methods of purification and preparation of lymphocytes used in these studies

would be anticipated to remove serum antibodies as well as usual varieties of

cytophilic antibodies.24 A possibility which requires consideration, however,

is that synthesis of antibodies to intrinsic factor may have occurred in vitro.

Alternatively there may be fundamental differences in the nature and develop-

ment of the immune responses expressed by those patients with either lympho-

cyte transformation or humoral antibodies to intrinsic factor.

The precise nature of the antigen, or antigens, responsible for eliciting

transformation responses by the lymphocytes of certain of these patients with

pernicious anemia cannot be deduced from these experiments. It is interesting

to note that the proportion of patients with pernicious anemia whose lympho-

cytes exhibited transformation in the presence of the various gastric antigen

preparations paralleled the intrinsic factor content of the lymphocyte cultures





( Table 3 ) . Caution must be expressed in concluding that these data indicate

that IF is the antigen responsible for evoking lymphocyte transformation

observed in these patients with pernicious anemia. It is conceivable that other

antigen( s) could have been purified in parallel with IF which although it was

Table 3.-Incidence of Lymphocyte Transformation of Pernicious Anemia PatientsWith Various Gastric Antigen Preparations

I.F. Binding Capacityof Vitamin B12

% of Pernicious AnemiaPatients Whose

Antigen PreparationIncluded in Each

Lymphocyte CultureLymphocytes Undersvent

Transformation

Gastric mucosal hoiiiogenate 3 ng. 3.4 (1/29)

Gastric juice 6 ng. 6.9 (2/29)

Purified intrinsic factor 30 ng. 21.4 (3/14)Concentrated gastric juice 36 ng. 37.5 (6/16)

(long-term cultures)

purified >200 fold in respect to protein concentration is not a completely

chemically pure preparation.

These experimental data, indicating transformation of lymphocytes from

certain patients with pernicious anemia cultures in the presence of a variety

of gastric antigens, permit the consideration that delayed hypersensitivity

mechanisms may be operative in pernicious anemia.

SUMMARY

Experiments were directed to the investigation of evidence of a possible

role of cellular immunity in patients with pernicious anemia. Lymphocyte-rich

peripheral leucocyte preparations from 29 patients with pernicious anemia

were cultured in the presence of a variety of preparations containing potential

antigens : these included human gastric juice, human intrinsic factor ( IF)

preparations, and human gastric mucosal homogenates. Lymphocyte trans-

formation was determined by measurement of the uptake of tritiated thymi-

dine into DNA. Lymphocyte transformation occurred when lymphocytes from

a portion of the patients with pernicious anemia ( 3.4 per cent to 37.5 per

cent ) were cultured in the presence of these antigen preparations. Lympho-

cyte transformation was noted in none of the patients whose sera contained

blocking antibodies to intrinsic factor. There was no correlation between

the presence or absence of lymphocyte transformation and the presence or

absence of serum antibodies to the gastric parietal cell cytoplasmic antigen.

These data on lymphocyte transformation permit the consideration that cel-

lular immunity may participate in the pathogenesis of pernicious anemia.

SUMMARIO IN INTERLINGUA

Experimentos esseva effectuate pro investigar Ic evidentia de un rob possibile de im-

munitate cellular in patientes con anemia perniciose. Preparationes de leucocytos peripheric

ric in lymphocytos ab 29 patientes con anemia perniciose esseva culturate in le presentia

de un varietate de preparationes continente antigenos potential. Istes includeva human

succo gastric, preparationes de human factor intrinsec, e homogenatos de human mucosa

gastric. Le transformation lymphocytic esseva determinate per mesurar le acceptation dethymidina a tritium ad in Ic acido deoxyribonucleari. Le transformation lymphocytic




70 TA! AND MCGUIGAN

occurreva quando lymphocytos ab un portion del patientes con anemia perniciose ( 3,4 pro

cento a 37,5 pro cento ) esseva culturate in le presentia de iste preparationes de antigeno.

Le transformation lymphocytic esseva notate in nulle del patientes qui habeva in br seros

anticorpore bloccante factor intrmnsec. Esseva constatate nulle correlation inter le presentia

0 absentia de transformation lymphocytic e le presentia o absentia de anticorpore seral antile antigeno cytoplasmic de cellulas parietal gastric. Iste datos relative al transformation

lymphocytic permitte le conclusion que immunitate cellular participa possibilemente in le

pathogenese de anemia perniciose.

REFERENCES

1. Taylor, K. B. : Immune phenomena

in pernicious anemia. Gastroenterology 45:

670, 1963.

2. Garrido-Pinson, G. C., Turner, M. D.,

Crookston, J. H., Samloff, I. M., Miller, L.

L., and Segal, H. L. : Studies of human in-

trinsic factor autoantibodies. J. Immun. 97:

879, 1966.

3. Fisher, J. NI., and Taylor, K. B. : A

comparison of autoimmune phenomena in

pernicious anemia and chronic atrophic gas-

tritis. New Eng. J. Med. 272:499, 1965.4. Samloff, I. M., and Barnett, E. V.:

Identification of intrinsic factor autoantibody

and intrinsic factor in man by radioim-

munodiffusion and radioimmunoelectropho-

resis. J. Immun. 95:536, 1965.

5. Irvine, \V. J., Davies, S. H., Dela-

more, I. W., and Wynn Williams, A. : Im-

munological relationships between pernicious

anemia and thyroid disease. Brit. Med. J. II:

454, 1962.

6. Taylor, K. B., Roitt, I. M., Doniach,

D., Couchman, K. G., and Shapland, C.:Autoimmune phenomena in pernicious ane-

mia: gastric antibodies. Brit. Med. J. II:1347, 1962.

7. Irvine, \V. J. : Gastric antibodies stud-

ied by fluorescence microscopy. Quart. J.

Exp. Physiol. 48:427, 1963.8. Jeffries, G. H. : Recovery of gastric

mucosal structure and function in pernicious

anemia during prednisolone therapy. Gas-troenterology 48:371, 1965.

9. Roitt, I. M., and Doniach, D. : De-

layed hypersensitivity in auto-immune dis-ease. Brit. Med. Bull. 23:66, 1967.

10. Broberger, 0., and Perlmann, P. : In

vitro studies of ulcerative colitis. I. Reac-

lions of patients’ serum with human fetal

colon cells in tissue cultures. J. Exp. Med.117:705, 1963.

11. Roitt, I. M., Jones, H. E. H., and

Doniach, D. : Mechanisms of tissue damage

in human and experimental autoimmune

thyroiditis. In Grabar, P. and Miescher, P.

A. (Eds. ) : Mechanisms of Cell and Tissue

Damage Produced by Immune Reactions,

2nd. International Symposium on Immuno-

pathology. Basel, Stuttgart, Benno Schwabe

& Co. 1964, p. 174.

12. Paterson, P. Y. : Transfer of allergic

encephalomyelitis in rats by means of lymphnode cells. J. Exp. Med. 111:119, 1960.

13. Hasting, J., Freedman, S., Rendon,

0., Cooper, H. L., and Hirschhorn, K.:

Culture of human white cells using differ-

ential leucocyte separation. Nature 192:

1214, 1961.

14. Dutton, R. W., and Eady, J. : An in

vitro system for the study of the mechanism

of antigenic stimulation in the secondary

response. Immunology 7:40, 1964.15. Robbins, J. H. : Tissue culture studies

of the human lymphocyte. Science 146:

1648, 1964.16. Mills, J. A. : The immunologic signif-

icance of antigen induced lymphocyte trans-

formation in vitro. J. Immun. 97:239, 1966.17. Nowell, P. C. : Phytohemagglutinin:

An initiator of mitosis in cultures of normal

human leukocytes. Cancer Res. 20:462,

1960.

18. Gottlieb, C., Lau, K., Wasserman, L.

R., and Herbert, V. : Rapic charcoal assayfor intrinsic factor ( IF), gastric juice un-saturated B52 binding capacity, antibody to

IF and serum unsaturated B1.� binding Ca-pacity. Blood 25:875, 1965.

19. Baur, S., Roitt, I. M., and Doniach,

D. : Characterization of the human gastric

parietal cell auto-antigen. Immunology 8:62, 1965.

20. Kabat, E. A., and Mayer, M. M.:

Experimental Immunochemistry ( ed. 2).

Springfield, Ill. Charles C Thomas, 1964,

p. 224.

21. Chosy, J. J., and Schilling, R. F.:Intrinsic factor studies: VII. The use of ion

exchange chromatography, gel filtration, and

ultrafiltration to purify the intrinsic factor

of human gastric juice. J. Lab Clin. Med.





61:907, 1963.22. Bray, G. A.: A simple efficient liquid

scintillatorfor counting aqueous solutions in

a liquid scintillation counter. Anal. Biochem.

1:279, 1960.23. David, J. R., Al-Askari, S., Lawrence,

H. S., and Thomas, L.: Delayed hyper-

sensitivity in vitro. I. The specificity of in-hibition of cell migration by antigens. J.Immun. 93:264, 1964.

24. Boyden, S. V. : Cytophilic antibody.

In Amos, B., and Kaprowski, H. (Eds.):Cell-Bound Antibodies. Philadelphia, The

Wistar Institute Press, 1963, p. 7.




1969 34: 63-71

CHIAKI TAI and JAMES E. MCGUIGAN Immunologic Studies in Pernicious Anemia

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