An antibody deficiency syndrome

An Antibody Deficiency Syndrome* Selective Immunoglobulin Deficiency with Reduced Synthesis of ry and Immunoglobulin Potypeptide Chains

a

WERNER F . BARTH, M .D ., RICHARD ASOFSKY, M .D ., THOMAS J . LIDDY, M .D ., Y . TANAKA, M .D ., DAVID S . ROWE, M .D .f and JOHN L . FAHEY, M .D .

Bethesda, Maryland HREE major classes of human serum immunoglobulins$ are distinguished by their antigenic, physical and chemical properties, i .e ., IgG (7Sy,-globulins), IgA (0 2,-globulins) and IgM (y t -nlacroglobulins) . In addition, a fourth class, designated IgD, has been described recently [7] . Immunoglobulin deficiency syndromes occur in several forms . Patients with agammaglobulinemia have a deficiency of all immunoglobulins [2,3] . This disease is characterized by recurrent infections and an impaired immune response after antigenic stimulation [4,5] . A similar clinical picture may be observed in some patients with selective immunoglobulin deficiencies in whom the serum levels of one or more classes of immunoglobulins are low and of the remaining immunoglobulins normal or high. Several types of selective immunoglobulin deficiency have been described . Single immunoglobulin deficiencies of IgA [6-17] or IgG [78] have been found . Multiple immunoglobulin deficiencies of two types have been recorded : (1) markedly decreased IgA and IgM with normal IgG [19-21] and (2) markedly decreased IgG and IgA with normal or increased 1gM [8,9,13,22-29] . The study of patients with selective immunoglobulin deficiencies offers a unique opportunity for investigating the function of individual im-

munoglobulins and the relationship between immunoglobulin deficiency and immune impairment . Two hypotheses have been proposed to account for the immunoglobulin deficiency syndromes . Rosen and Bougas [28] suggested that immunoglobulin deficiency diseases represent varying degrees and types of cellular maturation arrest . On the other hand, Fudenberg et al . [9] proposed that immunoglobulin deficiency can be explained by a genetically determined inability to synthesize selective components of the immunoglobulin molecule. Present evidence relating to the structure of immunoglobulin molecules indicates that these proteins are composed of two heavy and two light polypeptide chains [ .30,31 ] . The heavy chains of the immunoglobulin classes IgG, IgA, IgM and IgD (i .e ., the y, a, µ and S polypeptide chains) differ in physicochemical and antigenic properties and impart to the intact immunoglobulin molecule its class specificity [1,31,32] . The light chains are of two types, K (type t) and A (type it) [32-34] . The two light chains on a single molecule are either K or A (i .e ., type I or type if) . Thus a single immunoglobulin molecule has class specificity in its heavy chains (y, a, p or 6) and type specificity' in its light chains ( K or A, i.e ., type t or type ii) . We recently had the opportunity to study a patient with antibody and immunoglobulin deficiency syndrome who differed clinically in several respects from patients with this disorder described previously . There was no evidence of cellular deficiency or maturation arrest . Im-

I The terminology for immunoglobulins (gamma globulins) used in this paper was proposed by a committee sponsored by WHO and published in the Bull . World Health Organ., 30 : 447, 1964 .

* From the Immunology Branch and Metabolism Service, National Cancer Institute, the Germfree Laboratory, National Institute of Allergy and Infectious Diseases and the Pathologic Anatomy Branch, Clinical Center, National Institutes of Health . Department of Health, Education and Welfare . U . S . Public Health Service . Bethesda . Maryland . Manuscript received November 30, 1964 . f Research Fellow, Helen Hay Whitney Foundation . voI . 39 .

AUGUST 1965

319



320

Antibody Deficiency Syndrome-Barth et al .

munochemical, biochemical, morphologic and metabolic studies indicated that the production of y and a heavy polypeptide chains for the immunoglobulins in this patient was deficient .

MATERIALS AND METHODS Serum Protein and Immunoglobulin Studies . Serum paper electrophoresis, immunoelectrophoresis and analytical ultracentrifugation were performed using technics described previously [35] . Immunoelectrophoresis of the serum and urinary proteins was performed using specific antiserums against each of the immunoglobulin classes [35] . Quantitative immuneglobulin serum levels were determined using an antibody in agar gel diffusion technic [36] . Immunoglobulin Metabolism . Normal IgG globulin from a single sample of normal serum was isolated, labeled with radioactive iodine (1' 31 ) and studied in vim by standard procedures [37] . The IgM (y i -macroglobulin) was isolated from the patient's (C . C .) serum, labeled with 1 191 and studied metabolically by methods described previously [38] . Normal serum albumin was labeled with radioactive sodium chromate (COI) using the method described by Waldmann [39] . Morphologic Studies . Immunofluorescent studies of the patient's bone marrow, using specific antiserums prepared in rabbits against human immunoglobulins, were performed by methods described previously [40] . Chromosomal analysis of the bone marrow cells was performed (by Dr . J . Whang) using the method of Tjio and Whang [47] . Immunologic Studies. Bactericidal antibody titers were determined using the method of Landy and associates [42] . Titers to the Vi polysaccharide antigen of Escherichia coli and to a tularemia cell wall extract were performed using methods described perviously [43,44] . Type 0 red blood cells from a normal donor were labeled with Crn [45] and given in an injection to patient C . C . The survival of these cells was followed by counting the radioactivity in serial blood samples . Immunoglobulin Synthesis . Incorporation of C 14labeled isoleucine into the immunoglobulins was studied by combining roller tube cultures of bone marrow with immunoelectrophoresis of the culture fluids and autoradiography by the technic of Hochwald, Thorbecke and Asofsky [46] . Immunoglobulins were identified by a polyvalent antiserum as well as by specific antiserums reacting with 7, a, µ and S heavy polypeptide chains and with a and A (types i and n) light chains .

CASE REPORT The patient (C . C .) was a fifteen year old Navajo Indian girl who presented at the Clinical Center of the National Institutes of Health in June 1963 with

the chief complaint of recurrent infections and retarded growth . The patient's mother had been well throughout the pregnancy and delivery was spontaneous at term, the birth weight being 4 pounds 2 ounces . The patient had a mild infection in the upper respiratory tract at six months of age which required the attention of a physician . At two years of age she had a severe respiratory tract infection followed by bilateral cervical adenitis . Since that time she had multiple recurrent episodes of upper respiratory tract infections accompanied by fever and cervical adenitis . Between the ages of two and nine, incision and drainage of these cervical nodes were required on six separate occasions. The patient was admitted to another hospital at nine years of age for evaluation of cardiac enlargement and retarded growth . At that time she weighed 34 pounds and 3 ounces and her height was 41 inches, i .e ., she was at the three year old level for both height and weight . There was bilateral cervical adenopathy and a high-pitched cardiac murmur of grade 2 to 6 intensity along the left sternal border . Purulent material aspirated from a cervical lymph node on the left side was negative for acid-fast bacilli or fungus . Antibody deficiency was suspected at this time and the patient was started on gamma globulin therapy, 5 cc . being given intramuscularly each month . Between 1957 and 1963 the patient was given gamma globulin therapy sporadically. During this time she continued to have recurrent upper respiratory tract infections which were manifested by recurrent drainage from the left ear, intermittent sinusitis with nasal drainage, and recurrent cough . She also had measles, mumps and chickenpox with a normal rate of recovery . In early 1963 the patient had intermittent high fever that occurred several times a week . She also had mild to severe nasal bleeding with no apparent regularity . In March 1963 the patient presented at the U . S . Public Health Service Hospital, Shiprock, New Mexico, with complaints of fatigue and ankle swelling . There was generalized adenopathy, hepatosplenomegaly and cardiomegaly with a grade 4 to 6 systolic murmur at the apex . The hemoglobin was 4 .8 gm . per 100 ml ., the white blood cell count normal . The serum total protein was 5 .8 gm . per 100 ml . By paper electrophoresis serum albumin was 3 .1 gm . and gamma globulin 1 .0 gm . per 100 ml . A serum sample obtained at this time and tested in our laboratory indicated selective serum immunoglobulin deficiency (side infra) . The patient's mother is alive and well . The father has hepatic cirrhosis and esophageal varices . A brother (seventeen years old) and a half sister (fifteen months old) are alive and well . There is no family history of recurrent infections or diseases related to the clinical problems of the patient .

AMERICAN JOURNAL OP MEDICINE



Antibody Deficiency Syndrome-Barth et at . TAaLE I SERIAL SERUM PROTEIN AND IMMUNOGLORULIN LEVELS IN PATIENT

Date

t ei n s p o I (6 .0-8 .0 !gm .j10Dm1 .)

321

C. C.

IgG

IgA

(72)

(yIA$RA)

(y~M)

Globulin (9 .4-15 .4 mg . /ml .)

Globulin (1 .4-4.8 Mg . /ml . )

Globulin (0 .4-2 .0 Mg ./mi .)

0 .03 0

0 .8 1 .1 1 .0

0 .54 0 .16 0 .09 0 .08 0 .07 01

1 .1

0 .1

0

0 .52

0 .05

8 .1 14 .1 14 .0 14 .0 16 .2 13 .2 15 .0 10 .2

Albumin* y-Globulin • (0 .6-2 .0 (3 .1-5 .0 1 gm ./100m1 .)',gm ./f00ml .)

IgM

IgD Globulin (0 .003-0 .4 Mg,/MI .)

-4/8/63 1. 5/29/63t' 7/2/63 8/5/63 9/4/63 10/7/63 11/10/63 3/24/641

5 .8 7 .3 7 .0 6 .4 6 .7 6 .4 6 .7 6 .6

3 .1 3 .3 4 .1 3 .9 4 .1 3 .9

1 .0

-

0 0

0 0

0 .9 2 .0 1 .3 1 .0

1 .0 0 .6 3 .6

Figures in parentheses represent the normal levels, * Levels determined by paper electrophoresis. t Monthly injection of commercial -,-globulin discontinued . 1 Bimonthly injections of commercial y-globulin given since December 1963 .

NOTE :

On admission to the National Institutes of Health in June 1963 at the age of fourteen years ten months the patient's weight was 20 .7 kg. and height 122 .5 cm . (less than the third percentile for both) . Blood pressure was 100/60 mm . Hg and pulse 100 per minute . She was afebrile . There was no icterus, clubbing or cyanosis . A perforated tympanic membrane was present in the left ear . Dried blood was present in both nares . There was generalized adenopathy (cervical, axillary and inguinal) measuring 1 by 1 to 2 by 3 cm, Rhonchi and wheezes were present in the left hemithorax . The heart was slightly enlarged with a prominent pulmonic second sound and a grade 2 to 6 systolic murmur at the base . A prominent A wave was visible in the neck . Hepatosplenomegaly and edema were not present . There were no neurologic abnormalities . The hemoglobin was 9 .3 gm . per 100 ml . and the white blood cell count 9,900 per cu . mm ., with 71 per cent polymorphonuclear forms . The platelet count was 48,000 per cu . mm . The corrected erythrocyte sedimentation rate was 46 mm . per hour . Urinalysis did not reveal any abnormalities . Blood urea nitrogen, serum cholesterol, electrolytes, calcium, phosphorus, bilirubin and transaminase levels were all normal . Bromsulfalein retention at 45 minutes was 3 per cent and 2 per cent on two occasions . The results of the tests for syphilis, cold agglutinin, rheumatoid factor by bentonite flocculation, antinuclear antibodies and several lupus crythematosus cell preparations were all negative . Serum viscosity was 1 .4 and .5 1 on two occasions (normal 1 .4 to 1 .8) . The result of a cephalin flocculation test was 4+ and thymol turbidity was 16 units . These two abnormalities persisted and remained relatively unchanged during the VOL .

39, AUGUST 1965

observation period . Serum iron was 24, unbound ironbinding capacity 312 and total iron-binding capacity 336 jig . per 100 ml . The serum total protein was 7 .0 gm . per 100 mi . Albumin was 3 .3 and gamma globulin 1 .1 gm. per 100 ml . by paper electrophoretic analysis . (Tablet .) X-ray examination revealed chronic sinusitis with air-fluid levels in the maxillary sinus . Slight cardiac enlargement, predominantly right ventricular, was present on the basis of the electrocardiogram and x-ray studies. This was attributed to chronic lung disease, although bronchiectasis was not demonstrated . Bone age, determined from the x-ray studies, was equivalent to that at age ten or eleven years . Shortly after admission the patient became febrile with daily temperature spikes to 39 0 or 40 ° c . Salmonella paratyphi B was identified in the feces which also contained many Giardia lamblia organisms as well as pus and Charcot-Leyden crystals . The patient became afehrile two days after the start of chloramphenicol therapy which was continued for twelve days . Although the salmonella infection apparently was eliminated by the short course of chloramphenicol treatment, feral cultures again became positive for this organism approximately three weeks after administration of the drug was discontinued . The salmonella infection could not be eliminated despite intensive and long-term courses of amphicillin therapy alone, or in combination with gamma globulin, One episode of pneumonia occurred during the second month of hospitalization . Following discharge from the hospital the patient was given gamma globulin therapy at two to three week intervals . Frequent upper respiratory tract infections continued to occur despite replacement ther-

322

Antibody Deficiency Syndrome -Barlh et al .

utet Paper electrophoresis of the patient's serum with normal serum (1) compared with selective immunoglobulin deficiency (patient C. C .) (2) and typical agammaglobulinemic serum (3) . FIG . 1 .

FIG . 2 . Ultraecntrifugal analysis of patient's serum (above) contrasted with normal serum (below) . Sedimentation from left to right . Arrows indicate 18S peaks (left) and 6 .65 peaks (right) .

Immunoelectrophoretic comparison of patient's serum (right) with normal serum (left) using polyvalent antihuman immunoglobulin and specific anti-G serums for IgG (y2-globulin), IgA (y,_a or Ran) and 1¢M' (y,m or 6sm)x FIG . 3 .

apy, and examination in March 1964 disclosed the presence of pneumonia in the upper lobe of the right lung. RESULTS

Serum Immunoglobulins . Paper electrophoresis of the patient's serum revealed a normal protein pattern . (Fig . 1 .) Using this technic the gamma globulin fraction appeared to be normal, 15 per cent of the total serum proteins and 1 .0 gm . per 100 ml . Analytical ultracentrifugation of the

serum, however, demonstrated a marked increase in 18S globulins and virtual absence of 7S globulins . (Fig . 2 .) The immunoglobulins were investigated further, using immunoelectrophoresis to study individual classes of immunoglobulins . Immuneelectrophoresis using a polyvalent antiserum showed only one precipitin line in the patient's serum (Fig . 3) instead of the three precipitin arcs seen in normal serum . The observation that the specific antiserum reacted only with IgM AMERICAN JOURNAL OF MEDICINE

Antibody Deficiency Syndrome-Barth et al . globulins confirmed the impression that the major immunoglobulin in the patient's serum was IgM (18Sy,-macroglobulins) and indicated that this component was markedly increased . (Fig . 3 .) On immunoelectrophoresis of the patient's serum in the specific antiserums for IgG (7Sy,-globulin) and IgA (0,A-globulin), these inumunoglobulins were not detected . (Fig . 3 .) The results of quantitative immunochemical tests confirmed the very low serum levels of IgG and IgA . (Table i .) The IgG was detected initially in low amounts, but the patient had been receiving immunoglobulin therapy . The IgG level, however, showed a progressive fall over a six month period after exogenous globulin therapy was discontinued . The lowest level recorded was 7 mg . per 100 ml ., or approximately 0.5 per cent of normal . IgA was not detected in the patient's serum, although the method used can detect as little as 1 per cent of the normal value . The serum IgM was eight to fifteen times greater than the normal level during the period of observation, amounting to 1 .0 to 1 .5 gm . per cent. The level determined by the antibody agar plate technic was slightly higher than the total gamma globulin as estimated by paper electrophoresis, but this can be accounted for by the migration of some IgM globulins in the electrophoretic beta globulin region . A fourth class of serum immunoglobulin, IgD, usually is present in the serum of normal subjects but not in the serum of agammaglobulinemic subjects [1] . The levels of IgD globulin were markedly increased, however, in our patient's serum . (Table i .) The serum IgD level varied between 0 .6 and 3 .6 mg . per ml ., which is twenty to 120 times the median normal level. It is unlikely that this high level represents protein that the patient received exogenously, for it remained unusually high during a six month period in which no immunoglobulin therapy was given . Immunoglobulin Polyfertide Chains . Studies were undertaken to evaluate the heavy and light polypeptide chains of the immunoglobulins in the serum and urine of our patient . Purified IgM from the patient's serum was obtained by zone electrophoresis and filtration through G-200 Sephadex . The specific antigenic determinants characteristic of IgM were identified on the IgM globulins of patient C . C . by comparison with normal IgM on Ouchterlony analysis (Fig . 4) using rabbit antiserum prepared against normal IgM . VOL . . 39 . AUGUST 1965

323

Additional studies employing antiserum against the patient's IgM revealed a fusion of the precipitin lines formed by the patient's IgM and normal IgM in an Ouchtcrlony test in the same manner as shown in Figure 4 . Both type K and type L (types i and u) molecules were found in the IgM when it was tested with specific antiserums for type K and type L antigenic determinants . (Fig . 5, left .) Further confirmation was obtained by immunization of a rabbit with the patient's purified IgM . The rabbit antiserum contained antibodies directed against both type K and type L antigenic determinants . Thus the IgM from the patient was similar to normal IgM in that both type K and type L molecules were present . Immnmnoelectrophoretic studies of the patient's urine, concentrated thirty to fiftyfold by ultrafiltration, consistently showed a urinary globulin migrating in the gamma globulin region . On Ouchterlony analysis this urinary globulin gave a strong precipitin line with both antitype K and antitype L serums . (Fig . 5, nght .) These findings indicated the presence of K and L light polypeptidc chains in the urine . Larger imrnunoglobulins not detected as specific antiserums against IgG, IgA and IgM determinants failed to react with the urinary proteins . These immunochemical studies indicated that the patient was excreting abundant amounts of light chains of both antigenic types, unrnunoglobulin Metabolism . The metabolic conditions responsible for the observed protein abnormalities were studied using I'"-labeled normal IgG and I131-labeled autologous IgM . The catabolism of the I'"'-labeled normal IgG was found to be accelerated . (Table ii-) The observed half-time in patient C . C . was 15 .6 days, which is shorter than both the normal range of seventeen to thirty-two days [37J and the catabolic rate when an injection of the same preparation of I'"'-labeled IgG was given to two normal subjects (twenty and twenty-four days) and to a patient with agammaglobulinemia (fifty-six days) . The results of this additional test indicated that the labeled IgG preparation behaved as normal IgG [37] . The shortened survival of IgG in patient C . C . indicated that increased catabolism (or protein loss) contributed to the low serum levels . The patient was known to have salmonella enteritis and giardial infestation of the intestinal tract, which may have contributed to a transient, reversible protein-losing enteropathy . The low

324

Antibody Deficiency

Syndrome-Barth et at .

Ouchterlony analysis. Antihuman IgM antiserum reacted with purified preparation of normal human IgM (y,M) and patient's IgM (y,M-globulin). FIG. 4.

Ouchterlony analysis of antigenic properties of purified preparation of IgM from patient's serum reacted with antiA (I) and anti-L (a) antiserums (left) and concentrated preparation of patient's urine reacted with anti-K (I) and anti-L (a) antiserums (right) . FIG . 5 .

serum albumin levels (3 .1 and 3 .3 gm . per cent) prior to treatment for these infections, followed by a sustained increase after treatment (3 .9 to 4 .1 gm . per cent), are also consistent with this possibility . The presence of protein-losing enteropathy was confirmed by a Cry' albumin study performed immediately after the IgG turnover was completed . The fecal loss of Cry' during the first six days of the study was 0 .65 per cent of the administered dose . This figure was higher than the normal value (less than 0 .2 to 0 .4 per cent) [39] and confirmed the presence of a moderate degree of protein-losing enteropathy . Deficiency of IgG synthesis, however, appeared to be the major factor responsible for the low serum level . Calculation of the rate of IgG synthesis based on the lowest serum level and a half-time of fifteen and six-tenths days indicated that the patient may have been turning over 9 mg, per day . (Table n .) This calculated rate, which is about 1 or 2 per cent of the normal rate, probably is higher than the actual synthetic rate because the calculation was based on a measurement of serum IgG which may have included some IgG injected in an earlier treatment .

The metabolic behavior of I'M-labeled autologous IgM was studied after the salmonella infection was under control and no giardial organisms were demonstrated in the feces . The distribution and biological half-life were comparable to the results found for normal IgM . (Table n .) The synthetic rate was approximately ten times the mean synthetic rate found in normal subjects . The turnover studies, in summary, showed that the low serum IgG levels were the result of deficient synthesis and that the high IgM levels were due to increased synthesis . In addition, protein-losing enteropathy was demonstrated and was probably due to the associated salmonella and/or giardia infections. Morphologic Studies of Bone Marrow and Lymph Node Cells . The bone marrow was examined on

four different occasions . The only abnormalities were slight erythroid hyperplasia and lack of stainable iron . Lymphocytes, as well as plasma cells, were present in normal amounts . The number of plasma cells varied between 2 and 5 per cent of the marrow cells observed . The plasma cells present in the bone marrow had a normal AMERICAN JOURNAL OF MEDICINE



335

Antibody Deficiency Syndrome-Bar h et al . . u TAMS IMIUNOGLOBULIN METABOLISM

IgG l7S 7 2 ) Globulin Metabolism

IgM (y :) Macroglobulin Mctabolivu

Data

Plasma vol . (ml ./kg .) Serum level (mg . /ml .) Intravascular (per cent) . . Total amount in body (mg ./kg.) . .. . Half-life (days) Total body pool catabolized/day (%) Turnover rate (mg ./day) Turnover rate (mg ./kg ./day)

Normal Range [181 *

Patient

Normal

C. C.

Roote (79]"

Patient C. C.

35-52 6-16 32 51 600-1,700 17-32 2 .2-4 .1 870-3,710 16/51

48 0 .08 40 9 .6 15 .6 4 .4 10 .2 0 .43

27-59 06-2 .0 . 65-100 22-105 3 .8-6 .5 11 .8-18 .2 215-690 3 .4-17 .0

323 16 .2 `5 1,133 4 .6 15 .1 4,110 171 .3

`The normal values for IgG and IgM metabolism are derived from precious studies in this laboratory [19,36,37) . appearance on Giemsa stain (Fig . 6) and were periodic acid-Schiff (PAS)-negative . Chromosomal analysis of the cells in peripheral blood and bone marrow (performed by Dr . Jacqueline Whang) did not demonstrate any abnormalities . The lymph node architecture was normal grossly and a follicle was observed in the small specimen available . Plasma cells were abundant in the lymph node Some of the plasma cells were PAS-negative, whereas others were PASpositive . Electron microscopic examinations of the bone marrow and lymph node sections also demonstrated plasma cells . There was one such plasma cell with the dilated endoplasmic reticulum typically present in plasma cells and another cell with an intranuclear inclusion . (Fig . 7 .) There were no morphologic abnormalities in any of these cells other than the occasional intranuclear inclusion . Biosynthetic and Immunofluorescent Studies . Immunoglobulin synthesis in the patient's bone marrow was studied in vitro . Bone marrow cultures incorporated C 14-labeled amino acid into IgG, IgM and IgD immunoglobulins . (Fig . 8 .) The finding of labeling in the IgM and IgD molecules was not surprising in the light of the abundance of these molecules in the patient's serum, The presence of labeled IgG provided confirmation that some synthesis of this immunoglobulin was occurring in our patient . The antiserum used was specific for IgG as determined by other methods . VOL .

39,

AUGUST

1965

of twenty-one

control subjects

Immunofluorescent studies of the bone marrow were performed to identify the cells containing ihnmunoglobulins . Specific antiserums directed against IgG, IgA, IgM, and types K and L (t and u) molecules were used . Only a few cells in the marrow (less than one cell per several high power fields) fluoresced with the specific antiserum for IgG, a finding compatible with the markedly decreased synthesis of this protein . Bright fluorescence of many plasma cells was observed with the anti-IgM serum, however, and with the antiserums against types K and L light chains . (Fig . 9 .) The cells which fluoresced with these three antiserums appeared to be normal plasma cells . Some of the plasma cells in a section reacted with the anti-type K serum ; other plasma cells in the same section did not fluoresce . This was true using anti-type L, whereas almost all of the plasma cells in a section reacted with anti-IgM serum . This observation is compatible with other observations [47,48] indicating that K and A (type r and type n) light chains usually are present in different cells . IgA was found in small amounts after fractionation of patient C . C.'s serum permitted concentration of the IgA globulin so that it could be detected . The serum used for fractionation was obtained six months after the last injection of exogenous immunoglobulin . Although the biosynthetic and immunofluorescent studies did not detect IgA in the bone marrow, this immuneglobulin may have been formed elsewhere . In summary, these observations showed that

326

Antibody Deficiency Syndrome-Barth et al .

6

Fm . 6 . Bone marrow morphology in dysgammaglobulinemic patient (C . C .) . Arrows indicate plasma cells . Giemsa stain . Fin . 7 . Electron microscopic section of plasma cells in patient's lymph node . NUC = nucleus, ER = endoplasmic reticulum . M = mitochondria, INCL = intranuclear inclusion, MEM = nuclear membrane . Plasma cell on left is typical plasma cell with dilated endoplasmic reticulum . Plasma cell on right has intranuclear inclusion .

1 In vitro labeling of patient's immunoglobulins by bone marrow cells. Immunoelectrophoresis of normal and patient's serum is shown above . Autoradiograph of labeled protein is shown below . Unlabeled normal serum and patient's serum are used as carrier serums. Labeled IgM and JgG are shown. FIG . 8 .

the patient's plasma cells contained IgM and type K (t) and type L (n) immunoglobulins and, presumably, were synthesizing µ heavy polypeptide chains and K and X light polypeptide chains in abundant amounts . They also showed that some y (and possibly a) heavy polypeptide chains were being synthesized in small amounts . Serum Antibody Levels . Despite the presence of a Salmonella paratyphi B enteritis there were no agglutinating antibodies against this organism in the serum at any time during the patient's hospital course . Isohemagglutinins (anti-B) were not detected in the serum in many determinations over the six month observation period . Bactericidal antibodies were detected in the patient's serum (Table in) but not in the high titers often found in other patients [42] . Results of tests for cold agglutinins, rheumatoid factor and antinuclear antibody were all negative . Heterophil titers were low (1 :7) . Antibody Response After Antigenic Challenge . The patient had been given diphtheria toxoid in the past. The reaction to the Schick test performed shortly after admission was positive . Two injections of the diphtheria toxoid in alum were administered subcutaneously six weeks apart. Shortly after the second injection, the reaction AMERICAN JOURNAL OF MEDICINE



Antibody Deficiency Syndrome- Barth et al .

327

Type R (L)

Type UK)

FIG . 9 . Immunohuorescent studies of patient's bone marrow . Reaction with antiserums specific for p heave polypeptide chain, and type i and type n light chains are indicated . The three cells depicted are all plasma cells .

to a repeat Schick test was still positive, indicating an absence of antibody to the toxoid antigens . The Vi polysaccharide antigen of Esch . coli and a tularemia cell wall extract were also given . Despite three challenges with the Vi antigen (50 µg . given subcutaneously at one month intervals), no titers were demonstrated . Serum was examined at four to seven day intervals and more than fifteen determinations were all negative . Very low titers were detected with the tularemia antigen, but only after the second and third challenges . (Fig . 10 .) The response was markedly depressed as is shown by comparison with the response in eleven control subjects after only a single challenge . (Fig . 10 .) Antibodies :Red Blood Cell Antigen . The patient's serum did not contain any isohemagglutinins (anti-B) . The serum, however, did agglutinate type 0 red blood cells from ten different donors at low titer. This 0 cell agglutination was investigated further by following the survival of 5 ml . of intravenously administered Cr51 labeled 0 red blood cells (using the cells of a donor agglutinated by patient C. C .'s serum) . In patient C . C . the survival time of these cells was slightly shorter than normal during the first ten days of the study period . (Fig . 11 .) After ten days these cells were rapidly eliminated from circulation, which is compatible with the onset of immune elimination . Twenty-one days after the first challenge a second injection of cells voi . 39, AUGUST 1965

from the same donor resulted in prompt elimination of these cells from the circulation . The slightly shortened erythrocyte survival time observed during the first ten days following the initial injection may have been due to the coating of the 0 erythrocytes with the serum agglutinin demonstrated in vitro . The rapid elimination of the 0 cells ten days after the first injection and immediately after the second challenge suggests that the first injection of TABLE In A . Bactericidal Antibody Levels

Titer in I Patient C .C .-

Organism Esch . coli Sal. typhosa Aerobacter

Normal Titerf

100 32 1,000

320 1,320 320

B . Antibody Response in C. C .

Antigen Diphtheria toxoid . . . . Vi polysaccharide Tularemia

I

Challenges

2 3 3

Response Not detected Not detected Diminished

* Reciprocal of titer. f Normal values based on data of Drs . Weidanz and Landy, National Institute of Allergy and Infectious Diseases, National Institutes of Health .



328

Antibody Deficiency Syndrome-Earth et al .

12

I I Control subjeols

1

2

4

6

E

10

12

WEEKS AFTER INITIAL CHALLENGE

Antibody response to tularemia antigen . Response in eleven control subjects after single injection is shown . Antigen administered to patient at zero time, six weeks, and eleven weeks (arrows) . FIG . 10 .

labeled 0 cells had induced a primary immune response, separate from the factors involved in the in vitro agglutination reaction observed earlier . Delayed Hypersensitivity . Skin tests using intermediate purified protein derivative, coccidioidin, blastomycin and histoplasmin yielded negative results . Candida and trichophyton skin test antigens were also used, at dilutions ranging from 1 :300 to 1 :10,000 . There was no response to either of these antigens on two separate occasions . Eighty per cent of control subjects reacted positively to either, or both, of these skin test antigens at the dilutions used . These findings are compatible with the impairment of delayed hypersensitivity response in this patient . Family Studies . The immunoglobulin levels and isohemagglutinin titers were determined in members of the patient's family who were available for study . There was no immunoglobulin deficiency (Table iv) or clinical history of increased susceptibility to infection in the members studied .

while the remaining immunoglobulins are present in normal or increased amounts . An impaired immune response often accompanies selective immunoglobulin deficiency . Excluded are those immunoglobulin disorders associated with a malignancy of the lymphocyte-plasma cell system (multiple myeloma and macroglobulinemia) . Many forms of selective immunoglobulin deficiency occurring as single or multiple deficiencies have been described . Isolated deficiency of IgA (0 2A -globulin) is not rare and can occur in otherwise healthy subjects [12] . Immune impairment is usually not clinically evident [6-13] . Selective IgA deficiency has been found frequently in ataxia telangiectasia [14-17] . Immune impairment and repeated infections often occur in these patients . Selected deficiency of IgG (7Sy s-globulin) occurs and is accompanied by an antibody deficiency syndrome [18] . Isolated deficiency of IgM (y l-macroglobulins) has not been reported . Patients with one form of multiple immunoglobulin deficiency, i .e ., deficiencies of IgA and IgM with normal IgG levels, were described by Giedion and Scheidegger [19] . These patients had multiple infections and an impaired immune response after antigenic stimulation [19-21] .

0;

COMMENTS

The observations recorded in this paper have dual significance : clinical, with diagnostic and therapeutic implications for specific patients and immunogenetic, providing insight into the genes controlling immunoglobulin synthesis . Clinical . Selective immunoglobulin deficiency is characterized by a deficiency of one or more of the major forms of immunoglobulin

I I 5

10

IS

20

25

30

DAYS AFTER INJECTION

Survival of Crs1-Labeled 0+ red blood cells in patient . Second injection given twenty-one days after initial injection (arrow) . FIG . I1 .

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Antibody Deficiency Syndrome --Barth

329

el al .

TABLE IV LEVELS

OF IMMUNOGLOBULINS AND ISOHEMAGGLU'IININS IN 4AMIl

OF PATIENT C . C .

IgA (y,A) Globulin

lgM(1 i)

lgD

Globulin

Globulin

(9 .4-15 .4

(1 .4-4 .8

mg ./ml .)

mg ./mi .)

(0.4-2A mg ./ml .l

10 .003-0 .4 mg./ml .)

Mother Father *

15 .2 22 .0

3 .9 ' .1

Maternal halfsistcr . . . Brother Maternal aunt Maternal greatuncle . . . .

10 11 15 12

094 . 3 .0 3 .9 1 .9

JgG (y,)

Globulin

Members

.0 .5 .0 .0

0 .03 N .T . 1 0 1 1

.2 .84 .9 .2

0 .03 0 .15 N .T . NT .

Quantitative Isohemagglutinins (reciprocal of titer Anti-A, 64-512 : Anti-B, 32-256) Anti-B, 32 Anti-A, 256 Anti-B, 64 Anti-B, 8 Anti-B, 32 Anti-B, 256 Anti-B, 64

Figures in parentheses represent normal levels . N.T . = not tested . Had received blood transfusions before serum obtained . (Cirrhosis with bleeding esophageal varices .)

NorE :

Morphologic studies have not shown a consistent absence of plasma cells [20j . The second group of patients with multiple Immunoglobulin deficiencies are those with markedly decreased or virtually absent IgG and IgA and normal or increased IgM [8,9,13,2229] . Patient C . C. is in this general category . Both congenital and acquired forms have been reported . With one exception [27] all patients described have had repeated and significant clinical infections . The clinical, morphologic and immune findings are not uniform in patients with multiple immunoglobulin deficiencies . Plasma cells may be absent [22,27], present in small numbers [20,24,25], or present in relatively normal numbers (as in the present case) . In three patients described by Rosen et al . [23,28] plasma cells were not present but larger, primitive lymphocytoid plasma cells were found which contained IgM as detected by immunofluorescent technics [49] . Isohemagglutinins, bactericidal antibodies and heterophil antibody, all of which are predominantly IgM proteins, were present in high titers in some patients [23,28] . In our patient, however, titers against these antigens were either absent or present in the low normal range, despite the marked increase of IgM . Serum IgM levels can be normal or markedly increased, as in patient C . C . Thus a consistent morphologic, immunologic or clinical pattern has not been observed in patients with combined IgG and IgA deficiency . Selective immunoglobulin deficiency of IgG and IgA with normal or increased IgM may VOL .

39,

AUGUST

1965

occur more frequently than would be expected from the few cases reported in the literature . Immunoglobulin deficiency cannot be ruled out on the basis of a normal appearing serum paper electrophoresis, as shown in the present case . In addition, many patients thought to have generalized hypogammaglobulinemia may in fact have selective immunoglobulin deficiency . Soothill [501 studied serum IgM levels in seventy patients with hypogammaglobulinemia . In 10 per cent the serum IgM levels were two to five times greater than normal. These patients probably had selective immunoglobulin deficiency . The disorder may be suspected on clinical grounds in patients with recurrent infections who have either very high or absent isohemagglutinin titers and a decreased antibody response to antigenic stimulation . In these patients immunoelectrophoresis or quantitative studies of the serum immunoglobulin components are indicated to determine if selective immunoglobulin deficiency is present . The selective immunoglobulin deficiency syndrome must be distinguished from similar changes that may occur with lymphocytic or plasmocytic malignancy . Chronic lymphocytic leukemia, multiple myeloma and Waldenstrom's macroglobulinemia often are associated with generalized deficiency of all normal immunoglobulin components [51,52] and may involve a selective increase of one class of immunoglobulins . Myeloma proteins and macroglobulinemic macroglobulins, however, can be identified as either type K (t) or type L (it) molecules [53-55] . Thus the increase of only type i or type

330

Antibody Deficiency Syndrome- -Barth et al .

ii molecules is characteristic of malignant lymphocytic or plasmocytic disease and can be distinguished from the selective deficiency syndrome in which both types K (t) and L (u) IgM molecules are increased as in patient C .C . Furthermore, in patient C . C . the morphologic findings were not characteristic of lymphoma . The family data on serum immunoglobulin are insufficient to determine whether or not patient C . C . inherited the immunoglobulin defect . The fact that she had repeated infections from the age of two years would indicate that she had the defect at birth . Immunoglobulin (gamma globulin) replacement therapy may be given to patients with selective immunoglobulin deficiency in the same manner as in agammaglobulinemia . Prompt antibiotic treatment for infections and care to avoid infections are indicated . Eventually it may be possible to correct defects or to transfer normal immune cells in order to provide a fully competent immune system for these patients . Antibody Deficiency . Isolated IgA deficiency may not be associated with deficient immune response [6-13] . However, multiple immunoglobulin deficiencies, such as those found in patient C . C., usually are associated with antibody deficiency. The patients with IgG deficiency described by Rosen et al . [23] responded to antigenic challenge with IgM but not IgG antibodies . The patient described herein, however, had a poor or no response to almost all antigen tested, despite the high IgM levels and the lack of any cellular deficiency demonstrated by routine histologic technics . The immune impairment was not absolute, however, as shown by the response to Cr°n-labeled group 0 red blood cells . In patients with agammaglobulinemia the response to most antigens is reduced but not completely absent . In these patients delayed hypersensitivity can develop [29] and skin grafts be rejected [56] ; in some there is a response to bacterial and viral antigens [57,58] . The only patients whose clinical condition warrants suspicion of complete immunologic impairment are those with alymphocytosis and agammaglobulinemia [59-611 . These children rarely survive the first year of life . The poor antibody response in patient C . C . is partly understandable in terms of the deficiency of y and a heavy chains. The heavy chains are believed to play a key role in determining the antibody activity of immunoglobulin molecules

[62-67] . The presence of plentiful µ heavy chains, however, is contrasted with the absence of IgM antibodies . The immunochemical evidence indicated that the u chains were normal, but deficiency or abnormality of only a part of the chain may have escaped detection . Although aa defect causing impaired or altered synthesis of a key part of the antibody configuration of the immunoglobulin molecule could lead to the antibody deficiency observed, a defect interfering with the normal processes of response to antigen (prior to antibody synthesis) would have the same result . The frequency with which antibody deficiency accompanies selective irnmunoglobulin deficiency, however, is striking . Immunogenetic . The defect described here is characterized by deficient synthesis of the heavy polypeptide chains of IgG and IgA molecules (i .e ., y and a immunoglobulin polypeptide chains), superabundant synthesis of the heavy chains of IgM and IgD molecules (i .e ., u and 3 chains), and normal (or increased) synthesis of the K and X types (i and u) of light polypeptide chains . This defect may be considered in light of the concept that two types of genes, structural and regulator (controller), are responsible for normal protein synthesis [68] . If the structural genes for y and a polypeptide chains had been absent, neither of these chains would have formed . Biosynthetic studies, however, showed decreased synthesis of y heavy chains, and low levels of IgG and IgA were found in the serum six months after the last injection of exogenous immunoglobulin . Therefore we concluded that the structural genes for all the immunoglobulin polypeptide chains were present but some were functioning at a very low level . The failure to express normally the structural genes for y and a polypeptide chains could have been due to defects in the controller genes or to a failure occurring earlier in the mechanism of response to antigen . If the latter case were operative, the controller gene might not have been allowed to act . In either case the deficiency of two polypeptide chains (y and a), associated with an abundance of µ and S chains, may help to shed some light on the relative location of the structural genes responsible for the different heavy polypeptide chains . If the genes for y and a chains are adjacent (i .e ., without interposition of genes for µ and S chains) a single defect could account for the failure in normal expression of both genes for the y and a polypeptide chains, AMERICAN JOURNAL OF MEDICINE



Antibody Deficiency Syndrome ---Barth et al . i .e ., if the defect prevented "read off" of the two genes . Evidence that the genes regulating the y and a polypeptide chains may be linked comes from the work of Herzenberg [69] with mice . A parallel distribution of the separate isoantigens characteristic of IgG and IgA immunoglobulins was found in breeding experiments with mice. The genetic defect in patients with selective immunoglobulin deficiency is not sex-linked, for both female and male subjects are affected . A number of family studies have been reported and the genetic problems reviewed by Fudenberg, Ileremans and Franklin [9] and Burtin, Buffe and Grabar (13] . The latter investigators proposed that each heavy chain is controlled by a separate autosomal factor and that abnormalities in these factors are transmitted as recessive factors with the heterozygous state showing few or no abnormalities . Physiologic as well as genetic factors may play a role in the superabundance of IgM and IgD molecules (i .e ., µ and 5 chains) . Evidence was obtained in the present study indicating that the serum IgG level helped to determine the serum level of IgM . During two periods in which patient C . C. was receiving exogenous immunoglobulin (almost exclusively IgG) the serum IgM levels were 8 to 10 mg . per ml . During the six month study period in which no replacement therapy was given and the serum IgG level fell, the serum IgM levels rose to 13 to 16 mg . per ml . The serum IgM level fell to 10 mg . per ml . when immunoglobulin therapy was reinstituted . Soothill [50] and Huntley et al . [26] also described patients with this disorder in whom the serum IgM levels fell while the exogenous immunoglobulin therapy was being given . The lgG might regulate the synthesis of IgM molecules (or of µ polypeptide chains) by some homeostatic mechanism or by providing the specific antibody to reduce the stimulus to immunoglobulin formation . Alternatively, the exogenously supplied IgG may have altered µ heavy polypeptide chain synthesis through its effects on controller genes of the immunoglobulin synthetic process, analogous to the effects on controller genes proposed by Zuckerkandl 170] for the hemoglobin synthetic process . SUMMARY

Selective immunoglobulin deficiencies were identified in a fifteen year old Navajo Indian girl who had had repeated infections from infancy and retarded physical development . The

voi. .39 .

uc us i 1965

331

gamma globulins appeared to be normal on paper electrophoresis but immunoelectrophoresis revealed marked immunoglobulin abnormalities . The immunoglobulin abnormalities in this patient were marked reduction in the IgG (7Sy r globulins) and IgA (R2A-globulins), both less than one one-hundredth of normal ; and increased IgM (y i-macroglobulins) and IgD, both ten to fifteen times greater than normal . The ,naeroglobulins in this patient appeared to be normal in physiocochemical and immunechemical properties except for the low antibody content . Immune response was impaired, but not completely abolished . There were no cellular deficiencies or abnormalities in either the bone marrow or lymph nodes by routine staining technic . Well developed plasma cells were present in normal or increased numbers . Mature typical plasma cells and lymphoid-plasma cells demonstrated fluorescence with specific anti-IgM, anti-type K (t) and anti-type L (it) antiserums . The evidence presented shows that this patient had a defect in the synthesis of the y and a heavy polypeptide chains of the immunoglobulins (i .e ., those normally present in IgG and IgA molecules) . However, synthesis of µ and 6 heavy chains (of IgM and IgD molecules) was increased . Synthesis of K and X light chains (types i and u) probably was normal . The defective synthesis of y and a heavy polypeptide chains in this patient is compared with agammaglobulinemia and other selective immunoglohulin deficiency states . The structural genes for the y and a polypeptide chains were present in this patient . Evidence is presented indicating that defects in the controller or regulatory genes are a major feature of this disorder . Acknowledgment : We wish to thank Dr . S . J . Orlando of the U . S . Public Health Service Hospital, Shiprock, New Mexico, for bringing this case to our attention and for obtaining the serum samples from members of the patient's family . We also want to thank Dr . Maurice Landy, National Institute of Allergy and Infectious Diseases, for measurement of the bacteriocidal antibodies ; Dr . Paul Carbone, National Cancer Institute, for assistance in measuring the antibody levels and Vi and tularemia antigens ; Dr. Richard Malmgren, National Cancer Institute,



332

Antibody

Deficiency

for assistance with the immunofluorescent studies ; Dr . Robert Carpenter, National Institute of Allergy and Infectious Diseases, for determination of the fluorescent antinuclear antibodies ; and Dr . George Brecher and Dr . Paul Schmidt of the Clinical Center, for conducting the studies on antibodies reacting with group

0

erythrocytes and for assistance in the morphologic and isoagglutinin studies . REFERENCES 1 . RowE, D. S . and FAHEY, J . L . A new class of human immunoglobulin . Normal serum IgD. J. Exper . Med., in press. 2. SCHEIDEGGER, J . J . L'inmiuno-electrophorese . Semaine hop . Paris, 32 : 2119, 1956. 3 . GRABAR, P ., BURTIN, P . and SELIGMANN, M . Etudes immuno6lectrophoretiques et immunochimiques . Franc. etude c lin . des agammaglobulinbmies . Rev et biol., 3 : 41, 19584. GOOD, R . A . Studies on aganlmaglobulinemia and hypogammaglobulinemia . Proceedings of the First International Symposium on Immunopathology, pp. 41-59 . Basal, 1958 . 5 . GITLIN, D ., GRoss, P . A . M . and JANEWAY, C . A . The gamma globulins and their clinical significance. n . Hypogammaglobulinemia . New England J. Med ., 260 : 72, 1959 . 6 . HEREMANS, J . F . Les Globulines Serique du Systhme Gamma, p . 279 . Paris, 1960 . Arscia. 7 . WEST, C . D ., HONG, R . and HOLLAND, N . H Immunoglobulin levels from the newborn period to adulthood and in immunoglobulin deficiency states . J. Clin . Invest ., 41 : 2054, 1962. 8 . FUDENEERG, H ., GERMAN, J . L . . In and KUNKEL . H . G . The occurrence of rheumatoid factor and other abnormalities in families of patients with agammaglobulinemia . Arthritis & Rheumat ., 5 : 565, 1962 . 9 . FUDENBERG, H . II ., HEREMANS. J . F . and FRANKLIN, F . C. A hypothesis for the genetic control of syn. Inst . Pasteur thesis of the gamma globulins . Ann Lille, 104 : 155, 1963 . 10 . SONNET, J ., BRISBURS, P . and GILLART, C . Hypogammaglobulinemia familiale et cirrhose chez on adolescent Banton . Rev- internal . hepatot ., 13 : 37, 1963 . 11 . LevsN, W. C ., RITZMANN, S . E ., HAGGARD, M . E ., GREGORY . R . F . and REINARZ, J . A . Selective A-Beta-2 A globulinemia . Clin . Res ., 11 : 294, 1963 . 12 . ROCKEY, J . H., H.ANSON, L . A ., HEREMANS, J . F . and KUNxEL, H . G . Beta-2A aglobulinemia in two healthy men . J. Lab . & Clin . Med., 63 : 205, 1964 . 13 . BURTIN, P ., BUFFE, D . and GRABAR, P . Les hypogamma-globulinemies atypiques . Ann . Inst. Pasteur Lille, 106, 519, 1964. 14. YOUNG, R . R ., ADSTEN, K . F. and MOSER, H . W . Abnormalities of serum YIA-globulin and ataxia telangiectasia . Medicine, 43 : 423, 1964 . 15 . PETERSON, R . D . A ., KELLY, W, D . and GOOD, R . A . Ataxiatelangiectasia . Its association with a defective thymus, immunological deficiency disease and malignancy . Lancet, 1 : 1189, 1964 .

Syndrome-Barth

et at .

16. FIREMAN, P., BOESMAN, M . and GITLIN, D . Ataxia telangiectasis : a dysgammaglobulinemia with deficient )IS (92A-globulin) . Lancet, 1 : 1193, t964 . 17 . THIEFFRY, ST ., ARTHUI5, M ., LICARDI, J . and LYON, G . L'ataxie-telangiectasie . Rev . neural., 105 : 390, 1961 . 18 . SELIGMAN, M . Personal communication . 19 . CIEnloN, A. and SCHEIDEGGER, J . J . Kongenitale Immunparese bei Fehlen spezifischer Qs-Globuline and quantitative nomalen y-Globulinen . Helvet. paediat . acts, 12 : 241, 1957 . 20. BARANDUN, S., COTTIER, H . and HASSIG, A . New aspects of agammaglobulinemia and antibody deficiency syndrome. In : Immunopathology, p . 60. Basel, 1958 . Benno Schwabe & Co . 21 . GILBERT, C . and HONG, R . Qualitative and quantitative immunoglobulin deficiency . Am . J. Med., 37 :602, 1964. 22 . ISRAEL-ASSELAIN, R., BURTIN, P . and CHEBAT, J . Un trouble biologique nouveau : l'agammaglobulinemie avec $s-macroglobulin€mie . Bull- et mem . Soc. med. loop . Paris, 76 : 519, 1960 . 23 . ROSEN, F . S ., KEVY, S . V., MERLER, E ., JANEWAY, C . A . and GITLIN, D . Recurrent bacterial infections and dysgammaglobulinemia : deficiency of 7S gamma-globulins in the presence of elevated 195 gamma-globulins . Report of two cases . Pediatrics, 28 : 182, 1961 . 24 . BURTIN, P . Un example d'sgammaglobulin6mia atypique (on cas de grande hypogam pmaglobulinemie avec augmentation de In $,-Inacroglobuline) . Rev. Franc . etud . clin et biol ., 6 : 286 1961 . 25 . HONG, R ., SCHUBERT, W . K ., PERRIN, E . V . and WEST, C . D . Antibody deficiency syndrome associated with beta-2 macroglobulinemia . J. Pediat ., 61 : 831, 1962 . 26 . IIUNTLEY, C . C ., LAFFERTY, J . W . and LYERLY, A . Antibody deficiency syndrome with increased ynt(P,M)-globulins and absent 7LA(02A)-globulins and y,(G) globulins . Pediatrics, 32 : 407, 1963. 27 . HINZ, C . F ., JR . and BOYER, J . T. Dysgammaglobulinemia in the adult manifested as autoimmune hemolytic anemia : serologic and immunochemical characterization of an antibody of unusual specificity . New England J. Med., 269 : 1329, 1963 . 28 . ROSEN, F . S . and BOUGAS, J. A . Acquired dysgammaglobulinemia : elevation of the 19S gamma globulin and deficiency of the 7S gamma globulin in a woman with chronic progressive bronchiectasis . New England J. Med ., 269 : 1336, 1963 . 29 . Goon, R . A ., KELLY, W. D . and CABRIELSEN, A . E . Studies of the immunologic deficiency diseases : agammaglobulinemia, Hodgkin's disease and sarcoidosis . In : Proceedings of the 2nd International Immunopathology Symposium, p . 353 . Edited by Grabar, P . and Miescher, P . Basel, 1962 . Benno Schwabe & Co . 30. PORTER, R . R . The structure of gamma-globulin and antibodies . In : Basic Problems of Neoplastic Disease, p. 117 . Edited by Gelhorn, A . and Hirschberg, E . New York, 1962 . Columbia University Press . 31 . EDELMAN, G . M . and BENACERRAF, B . On structural and functional relations between antibodies AMERICAN JOURNAL OF MEDICINE



Antibody Deficiency Syndrome-Baelh et al . and proteins of the gamma-system . Proc . Nat . Acad. Se, 48 : 1035, 1962 . 32 . F .AHEV, J, L. Heterogeneity of y-globulin . Adr. Immune! ., 2 ; 41, 1962 . 33 . AIANNIx, M . and KUNKEL, H . G . Two major types of normal 7Sy-globulin . J. Exper. Med ., 117 : 213, 1963 . 34 . FxHEY . .r . L . Structural basis for the differences between Type i and Type a human -r-globulin molecules, J. Immune!., 91 : 448, 1963 . 35 . FAHEY, J . I . . and MCLAUGHLIN, C . Preparation of antisera specific for 6 .65 -y-globulin, OsA-globulins, y,-macroglobulins and for Type I and n common y-globulin determinants . J . Inunune!., 91 : 484, 1963 . 36 . FAHEY, J . L . and MOKELVEY, E . M . Quantitative determination of serum immunoglobulins using antibody in agar plates . J . Immune! ., 94 : 84, 1965 . 37 . SotoMON, A., WALDMANN, T . A . and FAHEY, J . L. Metabolism of normal 6 .6Sy-globulin in normal subjects and in patients with macroglobulinemia and multiple myeloma . J. Lab . & Clin. Med., 62 ; 1, 1963, 38 . BARTH, W . F ., WOCHNER, R. D ., WALDMANN, T . A . and FAHEY, J. L . Metabolism of human gamma macroglobulins . J. Clin . Invest ., 43 : 1036, 1964. 39, WALDMANN, T . A . Gastrointestinal protein loss demonstrated by >'Cr-labelled albumin . Lancet, 2 : 121, 1961 . 40 . SOLOMON, A., FAHEY, J . L . and MALMGREN, R . A . Immunohistologic localization of y,-macroglobulins, $2A-myeloma proteins, 6 .6Sy-myeloma proteins and Bence Jones protein . Blood, 21 : 403, 1963 . 41 . Tjro, L II . and WHANG, J. Chromosome preparation of bone marrow cells without prior in vitro culture or in vivo colchicine administration . Stain Technol., 37 ; 17, 1962 . 42. MICHAEL . J . G ., WHITBY, J . L . and LANDY, M . Studies on natural antibodies to gram-negative bacteria . J, Exper . Med ., 115 ; 131, 1962 . 43, LANDV, M . and LAMB, E . Estimation of Vi antibody employing erythrocytes treated with purified antigen . Proc. Soc . Exper . Biol . & Med., 82 : 593, 1953 . 44 . WOLFF, S . M ., WARD, S . B . and LANDY, M . Serologic properties of bentonite particles coated with microbial polysaccharides . Proc. Soc . Exper . Biol. & Med., 114 : 530, 1963 . 45 . READ, R . C . Studies of red-cell volume and turnover using radiochromium : description of new "closed" method of red-cell-volume measurement. New England J. Med., 250 : 1021, 1954 . 46 . HocawALD, G . M ., THORRECKE, G . J . and ASOFSKY, R . Sites of formation of immune globulins and of a component of C's . I . A new technique for the demonstration of the synthesis of individual serum proteins by tissues in vitro . J. Exper . Med ., 114 : 459, 1961 . 47, BENILR, G . M . and CERBRA, J . J . Polypeptide chains of human gamma-globulin . Cellular localization by fluorescent antibody. Science, 144 : 1590, 1964 . 48 . PERMS, B . and CHIAPPINO, G . Identification in human lymphoid tissues of cells that produce group I or group 2 gamma globulins . Immunology . 7 : 500 . 1964.

vi

vo1 .39, AGGUST 1965

333

49 . CRUCH .AUu, A., ROSEN, F . S ., CRAIG, J . M ., JANEWAY, C . A . and GITLIN, D . The site of synthesis of the 19S y-globulin in dysgammaglobulinemia . J. Exper . .14ed., 115 : 1141, 1962 . 50 . SOOTsILL, J . F . The concentration of y,-macroglobulin (Iota) in the serum of patients with hypogammaglohulinemia . Clin . Sc ., 23 : 27, 1962 . 51 . FAHEY . J . L ., SCOGCINs, R ., UTZ, J . P . and SZWED, C. F . Infection, antibody response and gamma globulin components in multiple myeloma and macroglobulinemia Ant J. Med. . 35 : 698, 1963 . 52 . McKELVEY, E . and FAHEY . J . L. Immunoglobulin changes in disease, in preparation . 53 . MANNIK, M . and KUNKEL, H . G_ Classification of myeloma proteins, Bencc Jones proteins and macroglobuhns into two groups on the basis of common antigenic characters . J. Exper. Med ., 116 : 859, 1962 . 54. FAHEY, J . L . and SOLOMON, A . Two types of ymyeloma proteins, 01A-myeloma proteins, y,macroglobulins and Bence Jones proteins identified by two groups of common antigenic determinants . J. Clin . Invest., 42 : 911, 1963 . 55 . KORNGOLD, L . and LIPARI, R . Multiple-myeloma proteins . L Immunological studies . Cancer, 9 : 183 . 1956 . 56 . SCHUBERT . W . K ., FOWLER, R ., JR ., MARTIN, L . W . and WEST, C . D . Homograft rejection in children with congenital immunological defects : agammaglobulinemia and Aldrich syndrome . Transplantation Bull ., 26 : 125, 1960 . 57 . MACCALLUM, F . 0 . Bacterial and viral antibodies in patients with hypogammaglohulinemia . Proc . Rov . Soc . Med . . 55 : 397, 1962 . 58 . B.ARNETT, E . V., NASOU, J . P ., UTz, J . P . and BARON, S. Antibodies to enteroviruses in hypogammaglobulinemic patients . New England J . Med ., 262 : 563, 1960 . 59 . Hrrzro . W . H . and WILLI, H . Hereditare tymphoplasmocytare Dysgenesie ("Alymphocytose mit Agammaglobulinamie") . Schweiz . med. Wchnschr ., 91 : 1625, 1961 . 60 . RosEN, F. S ., GITLIN, D . and JANEwAY, C . A . Alymphocytosis, agammaglobulinemia, homegrafts, and delayed hypersensitivity : study of a case . Lancet, 2 : 380, 1962 . (i1 . GrrLIN, D ., VAWTER, G . and CRAIG, J . M . Thymic alymphoptasia and congenital aleukocytosis . Pediatrics, 33 : 184, 1964. 62 . FLEISCHMAN, J . B ., PORTER, R . R . and PRESS . E . M . The arrangement of the peptide chains in yglobulin. Biochem . J., 88 : 220, 1963 . 63 . FRANEK . F . and NEzT IN, R . S . Recovery of antibody combining activity by interaction of different peptide chains isolated from purified horse antitoxins, Folio mioobial ., 8 : 128, 1963 . 7 64 . FRANEK, F ., KO'rYNEK, 0 ., SIMEK, L . and ZIKAN, J . S-sulphmlated antidinitrophenyl antibodies . In : Molecular and Cellular Basis of Antibody Formation, p_ 125 . Prague, 1964 . Publishing House of the Czechoslovakian Academy of Sciences . 65. EDELMAN, G . M., OLINS, D . I,., GALLY, J . A. and JINDER, N . D. Reconstitution of immunologic activity by interaction of polypeptide chains of antibodies . Proc . Nat . Acad . Sc., 50 : 753, 1963 . 66, FOVGEREAV, M ., OLINS, I) . E, and EDELMAN, G . M.



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Reconstitution of antiphage antibodies from L and H polypeptide chains and their formation of interspecies molecular hybrids . .1. Exper . Med., 120 : 349, 1964. 67 . ROHOLT, 0 ., ONOuE, K. and PRESSMAN, 1) . Specific combination of H and L chains of rabbit yglobulins . Proc . Nat . Aead. Sr ., 51 : 173, 1964 . 68 . JACOB, F . and MONOD, J . Genetic regulatory mecha-

nisms in the synthesis of proteins . J . Motic . Biol ., 3 : 318 . 1961 . 69 . HERZENeERG, L . A . A chromosome region for gamma-2A and beta-2A globulin H chain isoantigens in the mouse. Cold Spring Harbor Syroposta Quant. Biol . 24 : 455, 1964 . 70. ZUCRERRANOL, E . Controller gene diseases . J. Molec . Biol ., 8 :128, 1964 .

AMERICAN JOURNAL OF MEDICINE