April 1975
548
The Journal o f P E D I A T R I C S
Constrictive pericarditis, intestinal lymphangiectasia, and reversible immunologic deficiency A patient with constrictivepericarditis, secondary intestinal lymphangiectasia, andprotein-losing enteropathy was demonstrated to have the characteristic immunologic deficiency associated with intestinal lymphangiectasia: hypogammaglobulinemia, lymphocytopenia, cutaneous anergy, impaired allograft rejection, and diminished in vitro lymphocyte proliferative responses. Following surgical correction of the cardiac abnormality, the intestinal lymphangiectasia and protein-losing gastroenteropathy resolved and was accompanied by a slow but progressive return of normal immune function. This documented reversal of the immunologic deficiency in intestinal lymphangiectasia supports the concept that the immune defect in this syndrome is due to the excessive loss of lymphocytes and immunoglobulins into the gastrointestinal tract.
David L. Nelson, M.D.,* R. Michael Blaese, M.D., Warren Strober, M.D.,
Robert M. Bruce, M.D., and Thomas A. Waldmann, M.D., Bethesda, Md.
INTESTINAL LYMPHANGIECTASIA iS a primary or secondary disorder of the gastrointestinal tract associated with lymphatic dysfunction, bulk loss of lymph into the bowel lumen, and protein-losing enteropathy. 1 Previous studies have also demonstrated an associated i m m u n o l o g i c deficiency c o n s i s t i n g of h y p o g a m m a globulinemia, lymphocytopenia, cutaneous anergy, prolonged allograft survival, depressed in vitro lymphocyte proliferative responses to a variety of stimuli, and an increased incidence of neoplasia. 24 W e r e c e n t l y s t u d i e d a patient with c o n s t r i c t i v e pericarditis and secondary intestinal lymphangiectasia associated with immunologic deficiency. The gastrointestinal lymphatic dysfunction was potentially reversible by surgical correction of the cardiac disorder, and we r e a s o n e d that if the i m m u n o l o g i c d e f e c t also resolved we would have demonstrated that the immunologic deficiency in intestinal lymphangiectasia is attributable to the bulk loss of lymphatic fluid into the
From the Metabolism Branch~ National Cancer Institute, National Institutes o f Health. *Reprintaddress:Building 10, Room 4N117, National Institutes of Health, Bethesda, Md20014.
Vol. 86, No. 4, pp. 548-554
gastrointestinal tract. Our immunologic evaluations of this patient before and after successful pericardectomy support this hypothesis and form the basis of this report. Abbreviations used phytohemagglutinin Con A: concanavalin A PWM: pokeweed mitogen SF: staphylococcal filtrate SLO: streptolysin-O SKSD: streptokinase-streptodornase PPD: purified protein derivative PHA:
METHODS
Serum concentrations of immunoglobulins G, M, and A were determined by radial immunodiffusion in agar. 2 Serum IgE concentration was determined by doubleantibody radioimmunoassay. 5 A b s o l u t e l y m p h o c y t e c o u n t s o f p e r i p h e r a l blood were calculated from the total leukocyte count and the percentage,of lymphocytes seen on smear: each value represents the mean of at least three determinations. Cutaneous delayed hypersensitivity was assessed by injecting 0.1 ml of the following antigen solutions on
Volume 86 Number 4
Reversible immune deficit in lymphangiectasia
549
Fig, 1. Small intestinal biopsies performed A, before and B, after successful pericardectomy demonstrating reversal of intestinal lymphangiectasia. (• the volar surfaces of the forearms2: tetanus toxoid 10 L f / m l , , d i p h t h e r i a t o x o i d 10 L f / m l , i n t e r m e d i a t e strength PPD, m u m p s skin test antigen, and strept o k i n a s e - s t r e p t o d o r n a s e (40 to 10 u / m l ) . Reactions were read at 48 hours and considered positive if greater than 10 m m of induration was present. Assessment of sensitivity to dinitrochlorobenzene and the placement of a skin allograft from an unrelated v o l u n t e e r d o n o r were p e r f o r m e d as previously described. 2 Preoperatively, s e r u m a n t i b o d y titers were determined at weekly intervals following immunization with the following antigens: Brucella abortus, diphtheria toxoid, tetanus toxoid, and purified pneumococcal polysaccharide type I. Serum antibody titers to Brucella abortus were determined by microbacterial agglutination and titers for the other antigens, by micropassive hemagglutination. Proliferative responses of lymphocytes in vitro to the nonspecific mitogens phytohemagglutinin, concanavalin A, pokeweed mitogen, staphylococcal filtrate, and the specific m i t o g e n s streptolysin-O, s t r e p t o k i n a s e streptodornase, diphtheria toxoid, tetanus toxoid, candida, vaccinia~ and PPD were assessed by radioactive thymidine incorporation. 3 Results are expressed as a percentage of the patient's responses (mean of replicate samples) compared to those of two or more normal control subjects performed on the same day. Metabolic t u r n o v e r studies u s i n g purified radiolabeled albumin were performed by methods previously described) CASE REPORT
Patient R. B., a 15-year-old white male student from British Columbia, Canada, was admitted to the National Institutes of Health in November, 1971. He had been in good health until the summer of 1970, when he began having acute episodes of colicky abdominal pain followed by explosive diarrheal stools occurring four to six times a day. In February, 1971, abdominal
Fig. 2. Abdominal lymph node obtained prior to pericardectomy demonstrating depletion of thymus-dependent paracortical regions and preservation of germinal centers. (x56.) enlargement, dependent edema, and scrotal swelling were noted. About the same time, he had gradual onset of easy fatigability, dyspnea on exertion, and occasional sharp, nonradiating, anterior chest pain. In March, 1971, he was hospitalized in Kelowna, British Columbia, and found to have lymphocytopenia and hypoalbuminemia. Small bowel (Fig. 1, A) and lymph node (Fig. 2) biopsies were obtained andrevealed, respectively, dilated submucosal lymphatic vessels and paracortical lymphocytic depletion. A 51Cr-albumin stool clearance, performed in May, 1971, demonstrated 828 ml of plasma cleared into the stool daily (normal 5 to 24 ml/day). The diagnosis of intestinal lymphangiectasia with protein-losing enteropathy was made and dietary therapy was begun. The family history was negative for gastrointestinal disorders. Past infectious illnesses included uncomplicated mumps and rubella. The patient had received a basic series of DPT injections as an infant and a DT booster at age 14. He had been vaccinated for smallpox as an infant without difficulty. Physical examination at the time of admission revealed a chronically ill adolescent in moderate respiratory distress. Jugular venous distention to a height 0f 15 cm above the clavicles was present at an angle of 45 degrees with the horizontal, and prominent "a" waves were seen in the jugular venous pulsations. The point of maximal cardiac impulse was weakly
550
Nelson et aL
The Journal o f Pediatrics April 1975
Table I. Summary of albumin metabolism and serum immunoglobulin values Postoperative Preoperative
Plasma albumin concentration (gm/dl) 4-day 51Cr-albumin fecal excretion (% of injected dose) Gastrointestinal albumin loss (% of intravenous pool/day) Serum immunoglobulins IgG (mg/ml) IgA (mg/ml) IgM (mg/ml) IgE (ng/ml)
3 wk
6 mo
2.5
4.4
22
11.3
0.4*
17.4
--
5.0 0.97 0.42 86
5.2 1.0 0.72 --
1.85
18 mo
4.0 (3.5-4.6) (0-0.75)
.8 15.0 1.90 2.0 150
Normal mean (range)
0.58 (.1-1.7) 13.0 2.4 0.90 135
11.8 2.0 1.3 96
(9.5-14.6)t (1.16-3.4)]" (0.87-2.0)t (24-386)t
*3-Day excretion. ?Geometric mean (67%confidenceinterval.)
palpable in the left fifth intercostal space in the midclavicular line. A grade 2 of 6 nonradiating, midsystolic ejection murmur was audible at the lower left sternal border, accompanied by a high-pitched squeaky rub. The peripheral blood pressure was 110/60 mm Hg. A fluid wave was present on abdominal examination, and the scrotum was edematous. There was symmetric pitting edema to the midthighs bilaterally. Neurologic examination including visual field testing was normal. Admission laboratory data included a hemoglobin concentration of 14.6 gm/dl, a hematocrit of 45.2%, and a white blood Cell count of 5,200/mm3 with 75% polymorphonuclear cells, 14% lymphocytes, 7% monocytes, 3% eosinophils, and 1% basophils. Routine urinalysis was normal. Serum protein electrophoresis revealed a total protein of 3.0 gm/dl with albumin 1,3 gm/dl, al-globulin 0.3 gm/dl, c~2-globulin0.5 gm/dl, /3globulin 0.4 gm/dl, and y-globulin 0.5 gm/dl. Electrocardiogram was unremarkable except for uniformly low voltage in all leads. Upper gastrointestinal radiographs revealed an indistinct mucosal pattern compatible with mucosal edema. Small bilateral pleural effusions and diminished cardiac pulsations were observed by fluoroscopy. Thoracentesis yielded chylous fluid with 10,000 leukocytes/mm3, 5% polymorphonuclear cells; and 95% lymphocytes, a sugar concentration of 107 mg/ dl, and a protein concentration of 0.3 gin/all. The following pressures were obtained by cardiac catheterization: right atrium 20/19, right ventricle 32/20, pulmonary artery 32/20, left ventricle 85/23 (mm Hg). Marked thickening of the right heart border and severely compromised right ventricular diastolic expansion were seen on superior vena cava cinegram. On January 5, 1972, a pericardectomy was performed. Postoperatively, the patient required frequent infusions of albumin, diuretics, and digitalis for control of congestive heart failure and hypoproteinemia. At the time of discharge, three weeks postoperatively, he continued to have dyspnea on exertion and peripheral edema. The patient was readmitted in July, 1972, six months following pericardectomy. During this interval he had experienced gradual improvement in exercise tolerance and the peripheral edema had slowly resolved. A
peroral jejunal biopsy was performed, and re-evaluation of gastrointestinal protein loss and immune function was undertaken. His second postoperative admission was in May, 1973, eighteen months following pericardectomy. At this time he was in good health and had no edema or limitations of activity. General as well as cardiac examination results were normal. R E S U L T S OF S P E C I A L E V A L U A T I O N S
Preoperative (December, 1971). M e a s u r e m e n t s of albumin metabolism revealed an albumin half-life of 3.1 days ( n o r m a l 13 to 20), an a l b u m i n fractional catabolic rate of 25.4% of the intravenous pool per day (normal 9 to 13), reduced serum albumin concentration, and increased gastrointestinal albumin loss (Table I). As indicated by the SlCr-albumin stool clearance rate of 17% per day, the increased albumin fractional catabolic rate could be entirely accounted for by the excessive loss of protein into the gastrointestinal tract. Serum concentrations o f i m m u n o g l o b u l i n s G, M, and A were reduced, whereas the concentration of IgE was normal (Table I). Serum antibody titers following immunization were maximal at seven days and revealed the following rises in titers: t e t a n u s toxoid 4-fold, diphtheria toxoid 64-fold, Brucella abortus 512-fold, and p n e u m o c o c c a l polysaccharide I 2,048-fold. T h e absolute lymphocyte count was 642/mm 3, and cutaneous anergy to all five skin test antigens was observed (Fig. 3). Sensitivity to d i n i t r o c h l o r o b e n z e n e could n o t be d e m o n s t r a t e d , and a skin graft from an u n r e l a t e d volunteer was accepted without rejection. In vitro lymphocyte proliferative responses of peripheral blood l y m p h o c y t e s were 4 to 30% of s i m u l t a n e o u s l y performed c o n t r o l values for the nonspecific m i t o g e n s PHA, Con A, PWM, and SF and 4 to 22% of control values for the specific antigens SLO, SKSD, diphtheria
Volume 86 Number 4
Reversible immune deficit in lymphangiectasia
/ Absolute Lymphocyte Count
IO0
25oo
/
Vaccinia ~ . I ~
/
5 51
_o
200o 1500
"I"~
E
o -(
~>
I000 o
50
o
z 500
< _~ 2-~
o--~
t
I 3
I 6
PERICAROIECTOMY
SKIN TESTS NO. POSITIVE/NO. TESTED
0/5
I 9 MONTHS
I 12
I 15
2/5
I 18
3
I
4/5
Fig. 3. Changes in absolute lymphocyte count, in vitro lymphocyte proliferative responses, and skin test reactivity with pericardectorny.
toxoid, tetanus toxoid, candida, vaccinia, and PPD. Representative examples of proliferative responses to PHA and vaccinia are shown in Fig. 3. Studies of the PHA-induced proliferative responses of equal numbers of lymphocytes simultaneously obtained from pleural effusion and peripheral blood revealed a response of 114,067 dpm/5 x 105 in those from the pleural effusion and only 16,377 dpm/5 • 105 in those from peripheral blood. The proliferative response of the pleural effusion lymphocytes was within the range of response of normal peripheral blood lymphocytes. Three weeks postoperatively (January, 1972). At this time the serum albumin concentration had increased over the preoperative value, and the 4-day S]Cralbumin stool excretion was 11%, compared to 22% preoperatively (Table I). These findings are consistent with diminution o f gastrointestinal loss o f protein. Serum concentrations of immunoglobulins G, M, and A were still reduced (Table I). The skin graft remained in place without signs of rejection. The absolute lymphocyte count had risen to 1,593/mm3; however, in vitro l y m p h o c y t e proliferative responses remained markedly depressed with nonspecific mitogenic responses being 2 to 27% of normal values and specific mitogenic responses being 5 to 8% of simultaneously obtained normal values (Fig. 3). Six months postoperatively (July, 1972). The serum albumin concentration and stool excretion of 51Cralbumin were now normal, demonstrating reversal of the protein-losing enteropathy (Table I). A peroral jejunal biopsy (Fig. 1, B) revealed normal architecture with reversal of the intestinal lymphangiectasia. The absolute l y m p h o c y t e c o u n t was now 1,754/mm 3, a further increase over previous values, and cutaneous hypersensitivity to SKSD and mumps was now present
(Fig. 3). The skin allograft had been slowly rejected at home in May, 1972, four months after pericardectomy. In vitro lymphocyte proliferative responses were more nearly normal; nonspecific mitogenic responses were 60 to 100% of normal and specific mitogenic responses, 22 to 100% of normal (Fig. 3). Eighteen months postoperatively (May, 1973). The serum albumin concentration remained normal. Absolute lymphocyte count was 2,503/mm 3, and intradermal hypersensitivity to SKSD, mumps, diphtheria toxoid, and tetanus toxoid were all present (Fig. 3). An intermediate strength PPD skin test remained negative. Serum immunoglobulin concentrations remained normal (Table I). In vitro l y m p h o c y t e proliferative responses were now completely normal (Fig. 3). DISCUSSION I m m u n o l o g i c deficiency diseases of man usually result from decreased synthesis of immunocompetent cells and their products but also may result from increased catabolism of these immune effectors. 6 The diseases characterized chiefly by synthetic defects include the more familiar immunodeficiency diseases such as severe combined immunodeficiency, X-linked agammaglobulinemia, and isolated IgA deficiency. In these conditions there is presumably a block at some point in t h e pathway of differentiation of i m m u n o c o m p e t e n t cells. Although somewhat less common, a variety of diseases have been described in which increased catabolism of immune effectors occurs. Hypercatabolic immunologic defciency may be the result of either increased e n d o g e n o u s or exogenous (external losses) catabolism of immune effectors. Myotonic dystrophy is an example of the former mechanism in which isolated e n d o g e n o u s hypercatabolism of IgG results in de-
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creased serum IgG levels. 7 Intestinal lymphangiectasia with protein-losing enteropathy is an example of the latter mechanism in that reduced serum concentrations of immunoglobulins G, M, and A result from the loss of these proteins, along with other serum proteins, into the gastrointestinal tract. 2 In addition to these serum immunoglobulin deficiencies, patients with intestinal lymphangiectasia and protein-losing enteropathy have a severe cellular immune deficiency. 24 In previous studies, this cellular immune defect has been ascribed to lymphocytic depletion resulting from the bulk loss of lymphocytes from the recirculating lymphocyte pool into the gastrointestinal tract. 3Evidence for this hypothesis is as follows: (1) all of the protein-losing enteropathies associated with lymphocytopenia have demonstrable lymphatic abnormalities, 8-1~whereas the nonlymphocytopenic forms of gastrointestinal protein loss do not. 11"13(2) In the lymphocytopenic forms of protein-losing enteropathy, reversal of the protein loss also reverses the lymphocytopenia.9, 14, 15 (3) Lymphatic fluid containing radiopaque contrast media injected into the lymphatic vessels of the foot for lymphography has been shownto leak directly into the gastrointestinal tract in intestinal lymphangiectasia. 1618 (4) Loss of intravenously administered radiolabeled autologous lymphocytes into the gastrointestinal tract has been demonstrated in some patients with intestinal lymphangiectasia and pr0rein-losing enteropathy. 3 Lymphatic fluid in the thoracic duct has been demonstrated to contain several distinct populations oflymphocytesl In experimental animals, the majority of thoracic duct lymphocytes belong to a pool of thymusdependent,19, 20 long-lived cells which recirculate from peripheral blood to lymph via specialized postcapillary venules within peripheral lymphoid tissues121-23 Loss of lymphocytes normally present in thoracic duct lymph would be expected to result in deficiencies of immunologic function subserved by these cells. Prolonged external thoracic duct drainage in animals and man has been shown to result in peripheral blood lymphocytopenia, 24-28depletion of lymphocytes in thymusdependent regions of peripheral lymphoid tissues, 25, 27 cutaneous anergy, 25-27prolonged allograft survival, 26,28. 29and diminished in vitro proliferative responses of peripheral blood lymphocytes to PHA. z4, 30 In terms of cell-mediated i m m u n i t y , t h e r e f o r e , intestinal lymphangiectasia associated with protein-losing en, teropathy appears to be the clinical analogue of animals subjected to prolonged external thoracic duct drainage. However, it cannot be assumed that the increased catabolism of immune effectors is the sole or even chief
The Journal of Pediatrics April 1975
cause of the immunologic deficiency observed in these patients since an additional defect characterized by defective synthesis or differentiation of immunocompetent cells might be present. The patient described herein provided us with a unique opportunity to :examine these possibilities since the gastrointestinal lesion was secondary to constrictive pericarditis, a lesion potentially correctable by a surgical procedure without inherent effects on the immunologic system. On initial evaluation, this patient demonstrated all of the characteristic immune abnormalities associated with intestinal lymphangiectasia including hypogammaglobulinemia, lymphocytopenia, cutaneous anergy, delayed allograft rejection, and poor in vitro lymphocyte proliferative responses. In subsequent studies, cardiac function returned to normal following pericardectomy and concomitantly the structural and functional abnormalities of the gastrointestinal tract resolved, i.e., six months postoperatively both small bowel biopsy and tests for gastrointestinal protein loss showed no evidence of persistent disease. Associated with these improvements in cardiac and gastrointestinal lymphatic function was a return of normal immune function. This reversal of the previously documented immunodeficiency which occurred following normalization of cardiac and gastrointestinal lymphatic function strongly supports the concept that the immunodeficiency observed in patients with intestinal lymphangiectasia and protein-losing enteropathy is not due to defects in cellular differentiation or synthesis of immune effectors but rather to exogenous hypercatabolism of normal immune effectors within the gastrointestinal tract. From an immunologic viewpoint, several additional observations made'in the study of this patient are worthy of mention. The evaluation of cellular immune function revealed both quantitative and qualitative deftciencies in circulating peripheral blood lymphocytes, i.e., the patient had both lymphocytopenia and depressed lymphocyte proliferative responses in vitro when compared to control subjects in tests utilizing equal numbers of lymphocytes. This finding corroborates previous work in a larger group of similar patients 3 and may be explained by the fact that loss of lymphatic fluid and cells into the gastrointestinal tract, which is the equivalent of prolonged external thoracic duct drainage, would be expected to have its most pronounced effects on a population of lymphocytes which is both long lived and recirculates from blood to lymph and from lymph to blood. From the previously mentioned studies of experimental chronic drainage of the thoracic duct, it is deduced that this population of lyre-
Volume 86 Number 4
phocytes consists largely of t h y m u s - d e p e n d e n t cells which are necessary for in vitro lymphocyte proliferative responses. Thus both the quaIitative and quantitative deficiencies in circulating peripheral blood lymphocyte function in intestinal lymphangiectasia associated with protein-losing enteropathy are consistent with depletion of these long-lived recirculating cells through gastrointestinal losses. In the same way, the increased proliferative responsiveness of chylous effusion lymphocytes relative to peripheral blood lymphocytes in this patient would be expected by this hypothesis, since lymphocytes in chylous effusion would, by definition, be primarily recirculating lymphocytes. 3 in addition, it is of interest that the reversal of immunologic deficiency in this patient required a considerable amount of time. Six months postoperatively there was no evidence of ongoing hypercatabolism of imm u n e effectors, but qualitative and quantitative deficiencies of i m m u n e function persisted. This finding is consistent with the view that the replacement rate of the long-lived recirculating lymphocyte populations is quite slow, and many months must elapse before a full complement of i m m u n o c o m p e t e n t cells is again present in the peripheral blood and lymphoid tissues. This pattern is in marked contrast to the rapid cellular immunologic reconstitution following thymic transplants in the DiGeorge s y n d r o m e in which the t h e r a p e u t i c response probably involves expansion or terminal differentiation of an already existant cell population.31,32
REFERENCES 1.
2.
3.
4.
5.
6. 7.
8.
Waldmann TA: Protein-losing enteropathy, Gastroenterology 50:422, 1966. Strober W, Wochner RD, Carbone PP, et al: Intestinal lymphangiectasia: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytopenia and impaired homograft rejection, J Clin Invest 46:1643, 1968. Weiden PL, Blaese RM, Stroher W, et al: Impaired lymphocyte transformation in intestinal lymphangiectasia. Evidence for at least two functionallydistinct lymphocyte populations in man, J Clin Invest 51:1319, 1972. Waldmann TA, Strober W, and Blaese RM: Immunodeficiency disease and malignancy, Ann Int Med 77:605, 1972. Waldmann TA, Polmar SH, Balestra ST, et al: Immunoglobulin E in immunologic deficiency diseases, J Immunol 109:304, 1972. Strober W, Blaese RM, and Waldmann TA: Immunologic deficiency diseases, Bull Rheum Dis 22:686, 1972. Wochner RD, Drews G, Strober W, et al: Accelerated breakdown of immunoglobulin G (IgG) in myotonic dystrophy: a hereditary error of immunoglobulincatabolism, J Clin Invest 45:321, 1966. Petersen VP, and Hastrop J: Protein-losing enteropathy in
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constrictive pericarditis, Acta Med Scand 173:410, 1963. 9. Laster L, Waldmann TA, Fenster LF, et al: Albumin metabolism in patients with Whipple's disease, J Clin Invest 45:637, 1966. 10. Amman RW: Pathogenesis and etiology of regional enteritis, in Gastroenterology, Vol 2, Philadelphia, 1964, WB Saunders Company, pp 226-310. 11. Waldmann TA, Wochner RD, Laster L, et at: Allergic gastroenteropathy. A cause of excessive gastrointestinal protein loss, N Engl J Med 276:761, 1967. 12. Huizenga KA, Wollaeger EE, Green PA, et al: Serum globulin deficiencies in non-tropical sprue, with report of two cases of acquired agammaglobulinemia, Am J Med 31:572, 1961. 13. RUbin CE, Brandborg LL, Phelps PC, et al: Studies on celiac disease. I. The 'apparent identical and specific nature of the duodenal and proximal jejunal lesion in celiac disease and idiopathic sprue, Gastroenterology 38:28, 1960. 14. Strober W, Cohen LS, Waldmann TA, et al: Tricuspid regurgitation: a newly recognized cause of protein-losing enteroptahy, lymphocytopenia and immunologic deficiency, Am J Med 44:842, 1968. 15. Davidson JD, Waldmann TA, Goodman DS, et al: Protein-losing gastroenteropathy in congestive heart failure, Lancet 1:899, 1961. 16. Vesin P, Roberti A, Bismoth V, et al: Protein-and calcium-losing enteropathy with lymphatic fistula into the small intestine, in Physiology and pathophysiology of plasma protein metabolism, Stuttgart, 1965, Hans Huber, pp 179-185. 17. Stoelinga GBA, Van Monster PJJ, and Sloff JP: Chylous effusions into the intestine in a patient with protein-losing gastroenteropathy, Pediatrics 31:1011, 1963. 18. Mistilis SO, Skyring AP, and Stephen DD: Intestinal lymphangiectasia: mechanism of enteric loss of plasmaprotein and fat, Lancet 1:77, 1965. 19. Miller JFAP, Mitchell GF, and Weiss NS: Cellular basis of the immunological defects in thymectomized mice, Nature 214:992, 1967. 20. Miller JFAP, and Sprent J: Thymus-derived cells in mouse thoracic duct lymph, Nature (New Biol) 230:267, 1971. 2l. Gowans JL: The recirculation of lymphocytes from blood to lymph in the rat, J Physiol 146:44, 1959. 22. Marchesi VT, and Gowans JL: The migration of lymphocytes through the endothelium of venules in lymph nodes; and electron microscopic study, Proc R Soc Lond (Biol) 159:283, 1964. 23. Howard JC, Hout SV, and Gowans JL: Identification of marrow-derived and thymus-derived small lymphocytes in the lymphoid tissue and thoracic duct lymph of normal rats, J Exp Med 135:200, 1972. 24. Yu DTY, Peter JB, Stratton JA, et al: Lymphocyte dynamics: change in density profiles and response to phytohemagglutinin of human lymphocytes during prolonged thoracic duct drainage, Clin Immunol Immunopathol 1:456, 1973. 25. Fitts CT, Majeski JA, Sharbaugh RJ, et al: Immunosupression by thoracic duct filtration, Transplantation 14:236, 1972. 26. Tunner WS, Carbone PP, Blaylock WK, et al: Effect of
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thoracic duct drainage on the immune response in man, Surg Gynecol Obstet 121:334, 1965. 27. Searcy JR, and Fish TC: Immunological consequences of lymph lymphocyte depletion, Rev Surg 27:295, 1970. 28. Tilney NL, Atkinson JC, and Murray JE: The immunosuppressive effect of thoracic duct drainage in human kidney transplantation, Ann Intern Med 72:59, 1970. 29. McGregor DD, and Gowans JL: Survival of homografts of skin in rats depleted of lymphocytes by chronic drainage from the thoracic duct, Lancet 1:629, 1964.
The Journal of Pediatrics April 1975
30. Iverson JG: Phytohemagglutinin response of recirculating and non-recirculating rat lymphocytes, Exp Cell Res 56:219, 1969. 31. August CS, Rosen FS, Filler RM, et al: Implantation of a fetal thymus restoring immunological competence in a patient with thymic aplasia (DiGeorge's syndrome), Lancet 2:1210, 1968. 32. Steele RW, Limas C, Thurman GB, et al: Familial thymic aplasia. Attempted reconstitution with fetal thymus in a millipore diffusion chamber, N Engl J Med 287:787, 1972.
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The Journal o f P E D I A T R I C S
Constrictive pericarditis, intestinal lymphangiectasia, and reversible immunologic deficiency A patient with constrictivepericarditis, secondary intestinal lymphangiectasia, andprotein-losing enteropathy was demonstrated to have the characteristic immunologic deficiency associated with intestinal lymphangiectasia: hypogammaglobulinemia, lymphocytopenia, cutaneous anergy, impaired allograft rejection, and diminished in vitro lymphocyte proliferative responses. Following surgical correction of the cardiac abnormality, the intestinal lymphangiectasia and protein-losing gastroenteropathy resolved and was accompanied by a slow but progressive return of normal immune function. This documented reversal of the immunologic deficiency in intestinal lymphangiectasia supports the concept that the immune defect in this syndrome is due to the excessive loss of lymphocytes and immunoglobulins into the gastrointestinal tract.
David L. Nelson, M.D.,* R. Michael Blaese, M.D., Warren Strober, M.D.,
Robert M. Bruce, M.D., and Thomas A. Waldmann, M.D., Bethesda, Md.
INTESTINAL LYMPHANGIECTASIA iS a primary or secondary disorder of the gastrointestinal tract associated with lymphatic dysfunction, bulk loss of lymph into the bowel lumen, and protein-losing enteropathy. 1 Previous studies have also demonstrated an associated i m m u n o l o g i c deficiency c o n s i s t i n g of h y p o g a m m a globulinemia, lymphocytopenia, cutaneous anergy, prolonged allograft survival, depressed in vitro lymphocyte proliferative responses to a variety of stimuli, and an increased incidence of neoplasia. 24 W e r e c e n t l y s t u d i e d a patient with c o n s t r i c t i v e pericarditis and secondary intestinal lymphangiectasia associated with immunologic deficiency. The gastrointestinal lymphatic dysfunction was potentially reversible by surgical correction of the cardiac disorder, and we r e a s o n e d that if the i m m u n o l o g i c d e f e c t also resolved we would have demonstrated that the immunologic deficiency in intestinal lymphangiectasia is attributable to the bulk loss of lymphatic fluid into the
From the Metabolism Branch~ National Cancer Institute, National Institutes o f Health. *Reprintaddress:Building 10, Room 4N117, National Institutes of Health, Bethesda, Md20014.
Vol. 86, No. 4, pp. 548-554
gastrointestinal tract. Our immunologic evaluations of this patient before and after successful pericardectomy support this hypothesis and form the basis of this report. Abbreviations used phytohemagglutinin Con A: concanavalin A PWM: pokeweed mitogen SF: staphylococcal filtrate SLO: streptolysin-O SKSD: streptokinase-streptodornase PPD: purified protein derivative PHA:
METHODS
Serum concentrations of immunoglobulins G, M, and A were determined by radial immunodiffusion in agar. 2 Serum IgE concentration was determined by doubleantibody radioimmunoassay. 5 A b s o l u t e l y m p h o c y t e c o u n t s o f p e r i p h e r a l blood were calculated from the total leukocyte count and the percentage,of lymphocytes seen on smear: each value represents the mean of at least three determinations. Cutaneous delayed hypersensitivity was assessed by injecting 0.1 ml of the following antigen solutions on
Volume 86 Number 4
Reversible immune deficit in lymphangiectasia
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Fig, 1. Small intestinal biopsies performed A, before and B, after successful pericardectomy demonstrating reversal of intestinal lymphangiectasia. (• the volar surfaces of the forearms2: tetanus toxoid 10 L f / m l , , d i p h t h e r i a t o x o i d 10 L f / m l , i n t e r m e d i a t e strength PPD, m u m p s skin test antigen, and strept o k i n a s e - s t r e p t o d o r n a s e (40 to 10 u / m l ) . Reactions were read at 48 hours and considered positive if greater than 10 m m of induration was present. Assessment of sensitivity to dinitrochlorobenzene and the placement of a skin allograft from an unrelated v o l u n t e e r d o n o r were p e r f o r m e d as previously described. 2 Preoperatively, s e r u m a n t i b o d y titers were determined at weekly intervals following immunization with the following antigens: Brucella abortus, diphtheria toxoid, tetanus toxoid, and purified pneumococcal polysaccharide type I. Serum antibody titers to Brucella abortus were determined by microbacterial agglutination and titers for the other antigens, by micropassive hemagglutination. Proliferative responses of lymphocytes in vitro to the nonspecific mitogens phytohemagglutinin, concanavalin A, pokeweed mitogen, staphylococcal filtrate, and the specific m i t o g e n s streptolysin-O, s t r e p t o k i n a s e streptodornase, diphtheria toxoid, tetanus toxoid, candida, vaccinia~ and PPD were assessed by radioactive thymidine incorporation. 3 Results are expressed as a percentage of the patient's responses (mean of replicate samples) compared to those of two or more normal control subjects performed on the same day. Metabolic t u r n o v e r studies u s i n g purified radiolabeled albumin were performed by methods previously described) CASE REPORT
Patient R. B., a 15-year-old white male student from British Columbia, Canada, was admitted to the National Institutes of Health in November, 1971. He had been in good health until the summer of 1970, when he began having acute episodes of colicky abdominal pain followed by explosive diarrheal stools occurring four to six times a day. In February, 1971, abdominal
Fig. 2. Abdominal lymph node obtained prior to pericardectomy demonstrating depletion of thymus-dependent paracortical regions and preservation of germinal centers. (x56.) enlargement, dependent edema, and scrotal swelling were noted. About the same time, he had gradual onset of easy fatigability, dyspnea on exertion, and occasional sharp, nonradiating, anterior chest pain. In March, 1971, he was hospitalized in Kelowna, British Columbia, and found to have lymphocytopenia and hypoalbuminemia. Small bowel (Fig. 1, A) and lymph node (Fig. 2) biopsies were obtained andrevealed, respectively, dilated submucosal lymphatic vessels and paracortical lymphocytic depletion. A 51Cr-albumin stool clearance, performed in May, 1971, demonstrated 828 ml of plasma cleared into the stool daily (normal 5 to 24 ml/day). The diagnosis of intestinal lymphangiectasia with protein-losing enteropathy was made and dietary therapy was begun. The family history was negative for gastrointestinal disorders. Past infectious illnesses included uncomplicated mumps and rubella. The patient had received a basic series of DPT injections as an infant and a DT booster at age 14. He had been vaccinated for smallpox as an infant without difficulty. Physical examination at the time of admission revealed a chronically ill adolescent in moderate respiratory distress. Jugular venous distention to a height 0f 15 cm above the clavicles was present at an angle of 45 degrees with the horizontal, and prominent "a" waves were seen in the jugular venous pulsations. The point of maximal cardiac impulse was weakly
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Table I. Summary of albumin metabolism and serum immunoglobulin values Postoperative Preoperative
Plasma albumin concentration (gm/dl) 4-day 51Cr-albumin fecal excretion (% of injected dose) Gastrointestinal albumin loss (% of intravenous pool/day) Serum immunoglobulins IgG (mg/ml) IgA (mg/ml) IgM (mg/ml) IgE (ng/ml)
3 wk
6 mo
2.5
4.4
22
11.3
0.4*
17.4
--
5.0 0.97 0.42 86
5.2 1.0 0.72 --
1.85
18 mo
4.0 (3.5-4.6) (0-0.75)
.8 15.0 1.90 2.0 150
Normal mean (range)
0.58 (.1-1.7) 13.0 2.4 0.90 135
11.8 2.0 1.3 96
(9.5-14.6)t (1.16-3.4)]" (0.87-2.0)t (24-386)t
*3-Day excretion. ?Geometric mean (67%confidenceinterval.)
palpable in the left fifth intercostal space in the midclavicular line. A grade 2 of 6 nonradiating, midsystolic ejection murmur was audible at the lower left sternal border, accompanied by a high-pitched squeaky rub. The peripheral blood pressure was 110/60 mm Hg. A fluid wave was present on abdominal examination, and the scrotum was edematous. There was symmetric pitting edema to the midthighs bilaterally. Neurologic examination including visual field testing was normal. Admission laboratory data included a hemoglobin concentration of 14.6 gm/dl, a hematocrit of 45.2%, and a white blood Cell count of 5,200/mm3 with 75% polymorphonuclear cells, 14% lymphocytes, 7% monocytes, 3% eosinophils, and 1% basophils. Routine urinalysis was normal. Serum protein electrophoresis revealed a total protein of 3.0 gm/dl with albumin 1,3 gm/dl, al-globulin 0.3 gm/dl, c~2-globulin0.5 gm/dl, /3globulin 0.4 gm/dl, and y-globulin 0.5 gm/dl. Electrocardiogram was unremarkable except for uniformly low voltage in all leads. Upper gastrointestinal radiographs revealed an indistinct mucosal pattern compatible with mucosal edema. Small bilateral pleural effusions and diminished cardiac pulsations were observed by fluoroscopy. Thoracentesis yielded chylous fluid with 10,000 leukocytes/mm3, 5% polymorphonuclear cells; and 95% lymphocytes, a sugar concentration of 107 mg/ dl, and a protein concentration of 0.3 gin/all. The following pressures were obtained by cardiac catheterization: right atrium 20/19, right ventricle 32/20, pulmonary artery 32/20, left ventricle 85/23 (mm Hg). Marked thickening of the right heart border and severely compromised right ventricular diastolic expansion were seen on superior vena cava cinegram. On January 5, 1972, a pericardectomy was performed. Postoperatively, the patient required frequent infusions of albumin, diuretics, and digitalis for control of congestive heart failure and hypoproteinemia. At the time of discharge, three weeks postoperatively, he continued to have dyspnea on exertion and peripheral edema. The patient was readmitted in July, 1972, six months following pericardectomy. During this interval he had experienced gradual improvement in exercise tolerance and the peripheral edema had slowly resolved. A
peroral jejunal biopsy was performed, and re-evaluation of gastrointestinal protein loss and immune function was undertaken. His second postoperative admission was in May, 1973, eighteen months following pericardectomy. At this time he was in good health and had no edema or limitations of activity. General as well as cardiac examination results were normal. R E S U L T S OF S P E C I A L E V A L U A T I O N S
Preoperative (December, 1971). M e a s u r e m e n t s of albumin metabolism revealed an albumin half-life of 3.1 days ( n o r m a l 13 to 20), an a l b u m i n fractional catabolic rate of 25.4% of the intravenous pool per day (normal 9 to 13), reduced serum albumin concentration, and increased gastrointestinal albumin loss (Table I). As indicated by the SlCr-albumin stool clearance rate of 17% per day, the increased albumin fractional catabolic rate could be entirely accounted for by the excessive loss of protein into the gastrointestinal tract. Serum concentrations o f i m m u n o g l o b u l i n s G, M, and A were reduced, whereas the concentration of IgE was normal (Table I). Serum antibody titers following immunization were maximal at seven days and revealed the following rises in titers: t e t a n u s toxoid 4-fold, diphtheria toxoid 64-fold, Brucella abortus 512-fold, and p n e u m o c o c c a l polysaccharide I 2,048-fold. T h e absolute lymphocyte count was 642/mm 3, and cutaneous anergy to all five skin test antigens was observed (Fig. 3). Sensitivity to d i n i t r o c h l o r o b e n z e n e could n o t be d e m o n s t r a t e d , and a skin graft from an u n r e l a t e d volunteer was accepted without rejection. In vitro lymphocyte proliferative responses of peripheral blood l y m p h o c y t e s were 4 to 30% of s i m u l t a n e o u s l y performed c o n t r o l values for the nonspecific m i t o g e n s PHA, Con A, PWM, and SF and 4 to 22% of control values for the specific antigens SLO, SKSD, diphtheria
Volume 86 Number 4
Reversible immune deficit in lymphangiectasia
/ Absolute Lymphocyte Count
IO0
25oo
/
Vaccinia ~ . I ~
/
5 51
_o
200o 1500
"I"~
E
o -(
~>
I000 o
50
o
z 500
< _~ 2-~
o--~
t
I 3
I 6
PERICAROIECTOMY
SKIN TESTS NO. POSITIVE/NO. TESTED
0/5
I 9 MONTHS
I 12
I 15
2/5
I 18
3
I
4/5
Fig. 3. Changes in absolute lymphocyte count, in vitro lymphocyte proliferative responses, and skin test reactivity with pericardectorny.
toxoid, tetanus toxoid, candida, vaccinia, and PPD. Representative examples of proliferative responses to PHA and vaccinia are shown in Fig. 3. Studies of the PHA-induced proliferative responses of equal numbers of lymphocytes simultaneously obtained from pleural effusion and peripheral blood revealed a response of 114,067 dpm/5 x 105 in those from the pleural effusion and only 16,377 dpm/5 • 105 in those from peripheral blood. The proliferative response of the pleural effusion lymphocytes was within the range of response of normal peripheral blood lymphocytes. Three weeks postoperatively (January, 1972). At this time the serum albumin concentration had increased over the preoperative value, and the 4-day S]Cralbumin stool excretion was 11%, compared to 22% preoperatively (Table I). These findings are consistent with diminution o f gastrointestinal loss o f protein. Serum concentrations of immunoglobulins G, M, and A were still reduced (Table I). The skin graft remained in place without signs of rejection. The absolute lymphocyte count had risen to 1,593/mm3; however, in vitro l y m p h o c y t e proliferative responses remained markedly depressed with nonspecific mitogenic responses being 2 to 27% of normal values and specific mitogenic responses being 5 to 8% of simultaneously obtained normal values (Fig. 3). Six months postoperatively (July, 1972). The serum albumin concentration and stool excretion of 51Cralbumin were now normal, demonstrating reversal of the protein-losing enteropathy (Table I). A peroral jejunal biopsy (Fig. 1, B) revealed normal architecture with reversal of the intestinal lymphangiectasia. The absolute l y m p h o c y t e c o u n t was now 1,754/mm 3, a further increase over previous values, and cutaneous hypersensitivity to SKSD and mumps was now present
(Fig. 3). The skin allograft had been slowly rejected at home in May, 1972, four months after pericardectomy. In vitro lymphocyte proliferative responses were more nearly normal; nonspecific mitogenic responses were 60 to 100% of normal and specific mitogenic responses, 22 to 100% of normal (Fig. 3). Eighteen months postoperatively (May, 1973). The serum albumin concentration remained normal. Absolute lymphocyte count was 2,503/mm 3, and intradermal hypersensitivity to SKSD, mumps, diphtheria toxoid, and tetanus toxoid were all present (Fig. 3). An intermediate strength PPD skin test remained negative. Serum immunoglobulin concentrations remained normal (Table I). In vitro l y m p h o c y t e proliferative responses were now completely normal (Fig. 3). DISCUSSION I m m u n o l o g i c deficiency diseases of man usually result from decreased synthesis of immunocompetent cells and their products but also may result from increased catabolism of these immune effectors. 6 The diseases characterized chiefly by synthetic defects include the more familiar immunodeficiency diseases such as severe combined immunodeficiency, X-linked agammaglobulinemia, and isolated IgA deficiency. In these conditions there is presumably a block at some point in t h e pathway of differentiation of i m m u n o c o m p e t e n t cells. Although somewhat less common, a variety of diseases have been described in which increased catabolism of immune effectors occurs. Hypercatabolic immunologic defciency may be the result of either increased e n d o g e n o u s or exogenous (external losses) catabolism of immune effectors. Myotonic dystrophy is an example of the former mechanism in which isolated e n d o g e n o u s hypercatabolism of IgG results in de-
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creased serum IgG levels. 7 Intestinal lymphangiectasia with protein-losing enteropathy is an example of the latter mechanism in that reduced serum concentrations of immunoglobulins G, M, and A result from the loss of these proteins, along with other serum proteins, into the gastrointestinal tract. 2 In addition to these serum immunoglobulin deficiencies, patients with intestinal lymphangiectasia and protein-losing enteropathy have a severe cellular immune deficiency. 24 In previous studies, this cellular immune defect has been ascribed to lymphocytic depletion resulting from the bulk loss of lymphocytes from the recirculating lymphocyte pool into the gastrointestinal tract. 3Evidence for this hypothesis is as follows: (1) all of the protein-losing enteropathies associated with lymphocytopenia have demonstrable lymphatic abnormalities, 8-1~whereas the nonlymphocytopenic forms of gastrointestinal protein loss do not. 11"13(2) In the lymphocytopenic forms of protein-losing enteropathy, reversal of the protein loss also reverses the lymphocytopenia.9, 14, 15 (3) Lymphatic fluid containing radiopaque contrast media injected into the lymphatic vessels of the foot for lymphography has been shownto leak directly into the gastrointestinal tract in intestinal lymphangiectasia. 1618 (4) Loss of intravenously administered radiolabeled autologous lymphocytes into the gastrointestinal tract has been demonstrated in some patients with intestinal lymphangiectasia and pr0rein-losing enteropathy. 3 Lymphatic fluid in the thoracic duct has been demonstrated to contain several distinct populations oflymphocytesl In experimental animals, the majority of thoracic duct lymphocytes belong to a pool of thymusdependent,19, 20 long-lived cells which recirculate from peripheral blood to lymph via specialized postcapillary venules within peripheral lymphoid tissues121-23 Loss of lymphocytes normally present in thoracic duct lymph would be expected to result in deficiencies of immunologic function subserved by these cells. Prolonged external thoracic duct drainage in animals and man has been shown to result in peripheral blood lymphocytopenia, 24-28depletion of lymphocytes in thymusdependent regions of peripheral lymphoid tissues, 25, 27 cutaneous anergy, 25-27prolonged allograft survival, 26,28. 29and diminished in vitro proliferative responses of peripheral blood lymphocytes to PHA. z4, 30 In terms of cell-mediated i m m u n i t y , t h e r e f o r e , intestinal lymphangiectasia associated with protein-losing en, teropathy appears to be the clinical analogue of animals subjected to prolonged external thoracic duct drainage. However, it cannot be assumed that the increased catabolism of immune effectors is the sole or even chief
The Journal of Pediatrics April 1975
cause of the immunologic deficiency observed in these patients since an additional defect characterized by defective synthesis or differentiation of immunocompetent cells might be present. The patient described herein provided us with a unique opportunity to :examine these possibilities since the gastrointestinal lesion was secondary to constrictive pericarditis, a lesion potentially correctable by a surgical procedure without inherent effects on the immunologic system. On initial evaluation, this patient demonstrated all of the characteristic immune abnormalities associated with intestinal lymphangiectasia including hypogammaglobulinemia, lymphocytopenia, cutaneous anergy, delayed allograft rejection, and poor in vitro lymphocyte proliferative responses. In subsequent studies, cardiac function returned to normal following pericardectomy and concomitantly the structural and functional abnormalities of the gastrointestinal tract resolved, i.e., six months postoperatively both small bowel biopsy and tests for gastrointestinal protein loss showed no evidence of persistent disease. Associated with these improvements in cardiac and gastrointestinal lymphatic function was a return of normal immune function. This reversal of the previously documented immunodeficiency which occurred following normalization of cardiac and gastrointestinal lymphatic function strongly supports the concept that the immunodeficiency observed in patients with intestinal lymphangiectasia and protein-losing enteropathy is not due to defects in cellular differentiation or synthesis of immune effectors but rather to exogenous hypercatabolism of normal immune effectors within the gastrointestinal tract. From an immunologic viewpoint, several additional observations made'in the study of this patient are worthy of mention. The evaluation of cellular immune function revealed both quantitative and qualitative deftciencies in circulating peripheral blood lymphocytes, i.e., the patient had both lymphocytopenia and depressed lymphocyte proliferative responses in vitro when compared to control subjects in tests utilizing equal numbers of lymphocytes. This finding corroborates previous work in a larger group of similar patients 3 and may be explained by the fact that loss of lymphatic fluid and cells into the gastrointestinal tract, which is the equivalent of prolonged external thoracic duct drainage, would be expected to have its most pronounced effects on a population of lymphocytes which is both long lived and recirculates from blood to lymph and from lymph to blood. From the previously mentioned studies of experimental chronic drainage of the thoracic duct, it is deduced that this population of lyre-
Volume 86 Number 4
phocytes consists largely of t h y m u s - d e p e n d e n t cells which are necessary for in vitro lymphocyte proliferative responses. Thus both the quaIitative and quantitative deficiencies in circulating peripheral blood lymphocyte function in intestinal lymphangiectasia associated with protein-losing enteropathy are consistent with depletion of these long-lived recirculating cells through gastrointestinal losses. In the same way, the increased proliferative responsiveness of chylous effusion lymphocytes relative to peripheral blood lymphocytes in this patient would be expected by this hypothesis, since lymphocytes in chylous effusion would, by definition, be primarily recirculating lymphocytes. 3 in addition, it is of interest that the reversal of immunologic deficiency in this patient required a considerable amount of time. Six months postoperatively there was no evidence of ongoing hypercatabolism of imm u n e effectors, but qualitative and quantitative deficiencies of i m m u n e function persisted. This finding is consistent with the view that the replacement rate of the long-lived recirculating lymphocyte populations is quite slow, and many months must elapse before a full complement of i m m u n o c o m p e t e n t cells is again present in the peripheral blood and lymphoid tissues. This pattern is in marked contrast to the rapid cellular immunologic reconstitution following thymic transplants in the DiGeorge s y n d r o m e in which the t h e r a p e u t i c response probably involves expansion or terminal differentiation of an already existant cell population.31,32
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Waldmann TA: Protein-losing enteropathy, Gastroenterology 50:422, 1966. Strober W, Wochner RD, Carbone PP, et al: Intestinal lymphangiectasia: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytopenia and impaired homograft rejection, J Clin Invest 46:1643, 1968. Weiden PL, Blaese RM, Stroher W, et al: Impaired lymphocyte transformation in intestinal lymphangiectasia. Evidence for at least two functionallydistinct lymphocyte populations in man, J Clin Invest 51:1319, 1972. Waldmann TA, Strober W, and Blaese RM: Immunodeficiency disease and malignancy, Ann Int Med 77:605, 1972. Waldmann TA, Polmar SH, Balestra ST, et al: Immunoglobulin E in immunologic deficiency diseases, J Immunol 109:304, 1972. Strober W, Blaese RM, and Waldmann TA: Immunologic deficiency diseases, Bull Rheum Dis 22:686, 1972. Wochner RD, Drews G, Strober W, et al: Accelerated breakdown of immunoglobulin G (IgG) in myotonic dystrophy: a hereditary error of immunoglobulincatabolism, J Clin Invest 45:321, 1966. Petersen VP, and Hastrop J: Protein-losing enteropathy in
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constrictive pericarditis, Acta Med Scand 173:410, 1963. 9. Laster L, Waldmann TA, Fenster LF, et al: Albumin metabolism in patients with Whipple's disease, J Clin Invest 45:637, 1966. 10. Amman RW: Pathogenesis and etiology of regional enteritis, in Gastroenterology, Vol 2, Philadelphia, 1964, WB Saunders Company, pp 226-310. 11. Waldmann TA, Wochner RD, Laster L, et at: Allergic gastroenteropathy. A cause of excessive gastrointestinal protein loss, N Engl J Med 276:761, 1967. 12. Huizenga KA, Wollaeger EE, Green PA, et al: Serum globulin deficiencies in non-tropical sprue, with report of two cases of acquired agammaglobulinemia, Am J Med 31:572, 1961. 13. RUbin CE, Brandborg LL, Phelps PC, et al: Studies on celiac disease. I. The 'apparent identical and specific nature of the duodenal and proximal jejunal lesion in celiac disease and idiopathic sprue, Gastroenterology 38:28, 1960. 14. Strober W, Cohen LS, Waldmann TA, et al: Tricuspid regurgitation: a newly recognized cause of protein-losing enteroptahy, lymphocytopenia and immunologic deficiency, Am J Med 44:842, 1968. 15. Davidson JD, Waldmann TA, Goodman DS, et al: Protein-losing gastroenteropathy in congestive heart failure, Lancet 1:899, 1961. 16. Vesin P, Roberti A, Bismoth V, et al: Protein-and calcium-losing enteropathy with lymphatic fistula into the small intestine, in Physiology and pathophysiology of plasma protein metabolism, Stuttgart, 1965, Hans Huber, pp 179-185. 17. Stoelinga GBA, Van Monster PJJ, and Sloff JP: Chylous effusions into the intestine in a patient with protein-losing gastroenteropathy, Pediatrics 31:1011, 1963. 18. Mistilis SO, Skyring AP, and Stephen DD: Intestinal lymphangiectasia: mechanism of enteric loss of plasmaprotein and fat, Lancet 1:77, 1965. 19. Miller JFAP, Mitchell GF, and Weiss NS: Cellular basis of the immunological defects in thymectomized mice, Nature 214:992, 1967. 20. Miller JFAP, and Sprent J: Thymus-derived cells in mouse thoracic duct lymph, Nature (New Biol) 230:267, 1971. 2l. Gowans JL: The recirculation of lymphocytes from blood to lymph in the rat, J Physiol 146:44, 1959. 22. Marchesi VT, and Gowans JL: The migration of lymphocytes through the endothelium of venules in lymph nodes; and electron microscopic study, Proc R Soc Lond (Biol) 159:283, 1964. 23. Howard JC, Hout SV, and Gowans JL: Identification of marrow-derived and thymus-derived small lymphocytes in the lymphoid tissue and thoracic duct lymph of normal rats, J Exp Med 135:200, 1972. 24. Yu DTY, Peter JB, Stratton JA, et al: Lymphocyte dynamics: change in density profiles and response to phytohemagglutinin of human lymphocytes during prolonged thoracic duct drainage, Clin Immunol Immunopathol 1:456, 1973. 25. Fitts CT, Majeski JA, Sharbaugh RJ, et al: Immunosupression by thoracic duct filtration, Transplantation 14:236, 1972. 26. Tunner WS, Carbone PP, Blaylock WK, et al: Effect of
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thoracic duct drainage on the immune response in man, Surg Gynecol Obstet 121:334, 1965. 27. Searcy JR, and Fish TC: Immunological consequences of lymph lymphocyte depletion, Rev Surg 27:295, 1970. 28. Tilney NL, Atkinson JC, and Murray JE: The immunosuppressive effect of thoracic duct drainage in human kidney transplantation, Ann Intern Med 72:59, 1970. 29. McGregor DD, and Gowans JL: Survival of homografts of skin in rats depleted of lymphocytes by chronic drainage from the thoracic duct, Lancet 1:629, 1964.
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30. Iverson JG: Phytohemagglutinin response of recirculating and non-recirculating rat lymphocytes, Exp Cell Res 56:219, 1969. 31. August CS, Rosen FS, Filler RM, et al: Implantation of a fetal thymus restoring immunological competence in a patient with thymic aplasia (DiGeorge's syndrome), Lancet 2:1210, 1968. 32. Steele RW, Limas C, Thurman GB, et al: Familial thymic aplasia. Attempted reconstitution with fetal thymus in a millipore diffusion chamber, N Engl J Med 287:787, 1972.
