Clinical and immunologic observations in a patient with late onset immunodeficiency

GASTROENTEROLOGY

CASE

76:1458-1465,

1979

REPORTS

Clinical and Immunologic Observations a Patient with Late Onset Immunodeficiency

in

GUIDO N. TYTGAT, KEES HUIBREGTSE, PETER T. A. SCHELLEKENS, and THEA H. FELTKAMP-VROOM Division of Gastroenterology Gasthuis, Central Laboratory

Department

and Clinical Immunology, University of Amsterdam, Wilhelmina of the Netherlands Red Cross Blood Transfusion Service, and

of Pathology, Slotervaartziekenhuis,

The case of a patient with common variable immunodeficiency and duodenal nodular lymphoid hyperplasia, suffering from diarrhea and abdominal pain, is presented. lmmunoglobulins could not be detected or were very low in serum, tissues, and external secretions. Serum antibody production and delayedtype immunity were disturbed, but T-killer and Kcell function were normal. Cultured with pokeweek mitogen, the patient’s B-cells failed to synthesize cytoplasmic immunoglobulin when patient T-cells were present in the medium but not when normal T-cells were added. In addition, patient T-cells suppressed Ig production by control B-cells, which suggests that the patient T-cell population was primarfor the observed abnormal B-cell ily responsible function. As shown by positive reaction with a specific antihuman T-cell antiserum, the lymphoid cells in the lymphoid nodules in the gut wall consisted to a large extent of T-lymphocytes. Treatment with metronidazole for giardiasis, gammaglobulin, antibiotics, and antidiarrheals was without benefit. As a terminal event, the patient developed extensive ulcerative destruction of the distal small bowel associated with evidence of widespread cytomegalovirus infection. The CMV-cells were especially numerous in the severely ulcerated small bowel. It seems highly probable that the CMV-infection contributed Received September 5, 1978. Accepted January 24, 1979. Address requests for reprints to: Prof. Dr. Guido N. Tytgat, M.D., Division of Gastroenterology, University of Amsterdam, Wilhelmina Gasthuis, Eerste Helmersstraat 104,1054 EG Amsterdam, The Netherlands. The authors gratefully acknowledge the expert help of F. Gmelig-Myeling and A. LJytdenHaag, Division of Immunology, University Hospital, Utrecht, of Drs. D. Agenant, P. Elte, and R. Grijm, for patient care, and of Mr. G. Pfau and Mr. C. Gravemeijer, for technical assistance. 0 1979 by the American Gastroenterological Association OOlS-5065/79/061458-08$02.00

Amsterdam,

The Netherlands

to the symptom complex of intractable abdominal pain, diarrhea, protein loss, and malabsorption. The case is described of a patient with common variable hypogammaglobulinemia, associated with duodenal nodular lymphoid hyperplasia, who developed intractable abdominal pain and diarrhea, resistant to conventional therapeutic measures. Particular attention is paid especially to the characterization of the lymphocytes in the intestinal lymphoid nodules, to the suppressive effect of patient T-lymphocytes upon B-lymphocyte maturation, and to the development of extensive destructive ulceration of the small intestinal mucosal lining in conjunction with overwhelming cytomegalovirus infection.

Case Report E.P.A. was born in 1921, the product of a normal pregnancy. The neonatal period was uneventful. She was well and healthy until the age of 17, except for two episodes of bronchopneumonia during childhood. She married and had four normal children. She suffered from recurrent respiratory infections, mainly rhinitis and sinusitis, for about 12 yr, for which a bilateral CaldwellLuc operation was carried out at the age of 35. In 1974, she started complaining of intermittent attacks of watery nonbloody diarrhea lasting for a few days or weeks, preceded by periumbilical crampy pain. Gradually, the diarrhea became more severe, occurring about five times during the day and two to three times at night. In addition, she started complaining of nausea, anorexia, and progressive weight loss from 69 kg in 1971 to 50 kg in 1976. Because of the progressive nature of her complaints, she was medically investigated elsewhere. Radiologic examination of the stomach and small and large bowel was noncontributory. Oral and intravenous cholangiography failed to visualize the biliary system. Sigmoidoscopy and rectal

CASE

June 1979

biopsy showed a mildly inflamed mucosa, thought to be compatible with chronic active nonspecific colitis. Repeated fecal examination for the presence of pathogens, ova, or parasites was negative. At this point, the patient was hospitalized in our unit for further evaluation. Physical examination upon admission showed findings consistent with pulmonary emphysema. The liver edge was palpable 3 cm underneath the costal margin and the splenic tip was just palpable. Routine hematologic parameters were normal. Other relevant laboratory data are summarized in Table 1. Normal values were obtained for urinary indican and 5-hydroxy indole acetic acid. The 14Cglycocholate breath test did not reveal enhanced bacterial bile acid deconjugation. Quantitative aerobic and anaerobic cultures of fasting jejunal content were initially not indicative of bacterial overgrowth but became abnormal later on in the evolution (Table 1). Throughout the study there existed a moderate hypoproteinemia with a low albumin concentration and a profound hypoglobulinemia (Table 1). Radiologic examination showed innumerable tiny smooi.h nodular filling defects, some 3 mm in diameter, uniformly distributed over the duodenum. Gastroscopy showed obvious atrophic changes of the gastric mucosa without evidence of polypoid lesions. Duodenoscopy revealed numerous tiny sessile polypoid lesions which, together with the appearance on the x-ray, were highly suggestive for nodular lymphoid hyperplasia. Colonoscopy was interpreted as normal, but evidence of mild nonspecific mucosal inflammation was seen on multiple biopsies. A repeat intravenous cholangiogram did visualize the biliary system and revealed no evidence of biliary calculi.

Table

Z. Biochemical

Data of Patient

with Common

Immunologic

REPORTS

Studies

Detailed immunologic studies were performed in reference to both humoral and cell-mediated immunity. The total number of lymphocytes in the peripheral blood was normal at 2,000 per mm3. The percentage of T-lymphocytes, measured either with a T-cell antiserum or with the E-rosette technique, was normal, with a value of 84% and 74%, respectively, but the percentage of B-lymphocytes, measured by immunofluorescence, was only 4% (normal range 6-20X). The remainder of the lymphocytes lacked characteristics of both T- and B-cells. IgG, IgA, and IgM globulin levels were uniformly low or not detectable as summarized in Table 1. Complement levels C3 and C4 of 110% and 274%, respectively, were normal (normal range 65-170X). Salivary samples did contain free secretory piece but lacked secretory IgA. /%Isohemagglutinin was absent from the plasma. Antitetanus antibodies were lacking both before and after boosting with tetanus toxoid. Antibodies to influenza, measles, herpes simplex, and cytomegalovirus were undetectable upon repeated testing. Antibodies directed against IgA were not present. In addition, no antibodies were detectable against thyroid parenchyma and colloid, gastric parietal cells, smooth muscle cells, mitochondria, adrenal gland, skeletal muscle, and salivary gland. Rheumatoid serology was negative and the antistreptolysin titer was 100 U.

Variable Immunodeficiency Values on:

Normal values Body weight (kg) Hemoglobin (mmoJ/liter) 8.7-10.6 Leuk.ocytes x lO’/liter 4-10 Total protein (g/liter) 60-80 Albumin (g/liter) 35-50 Gamma globulin (g/liter) 8-16 IgG (IU/liter) 65-200 IgA (JU/liter) 40-225 IgM (W/liter) 45-335 Alkal. phosphatase (JIJ/liter) 20-60 11-32 Iron (pmol/Jiter) IBC bmol/liter) 45-72 Folic acid (nmol/liter) 7-35 B,2 (pmol/Iiter) ZOO-700 Carotene (pmol/liter) 1-3 Fecal fat (g/day) <5 Fecal plasma loss 2-23 (“‘Cr Cl,) (ml/day) Bacterial content jejunal fluid 110" Aerobes
lo/76 50 8.1 6.0 53 25 2.5 30 0 0 282 2.1 6.2 100

12/76

z/77

7/77

49

7.3 6.5 51 34 25 <5 5

1459

30 0 (5 124 9.0 50 8.9 598 0.42 9 94

>lo"


a/77 40

7.7 5.8 38 23

6.5 3.9 40 22

3 21 11

30 10 25

274 5.0

356 5.7

10.8 670 0.59 20

9/77 38 5.5 2.9 45 25

141 4.0

0.14

51 >lo” >lo”

10/77 36 5.2 3.0 47 29

91

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CASE REPORTS

Studies of delayed-type immunity by skin testing with varidase, trichophyton, candida, mumps and PPD antigens showed only reactivity toward candida antigen (wheel 15 x 15 mm). Upon DNCB sensitization and challenge, nonreactivity was observed. Abnormal T-lymphocyte function was also documented by studies of in vitro blast cell transformation. The results after stimulation with the nonspecific mitogen or T-lectin phytohemagglutinin and after stimulation in the mixed lymphocyte culture were 7% and 50%, respectively (normal values > 50%). However, testing for cell-mediated lympholysis (CML),’ a measure of T-killer cell function, was normal with a 47% lysis of the target cells of a normal unrelated individual. Testing for antibody-dependent cellular cytotoxicity (ADCC), a measure of K-cell function, was also normal, according to the criteria of Zeylemaker et al.’ Examination of multiple small bowel suction biopsy specimens obtained from the duodenum and proximal jejunum showed huge collections of lymphocytes, occasionally mixed in the center with larger germinal center-type cells. Ultrastructurally, the lymphoid nodules were made up of large cells closely adjacent to each other, showing large, somewhat irregular nucleoli and sparse cytoplasm, containing only a few mitochondria and some strands of endoplasmic reticulum, surrounded by abundant clusters of ribosomes. The mucosal architecture in between the lymphoid nodules varied from normal via all grades of abnormality to a flat avillous appearance. In addition, there was evidence of massive infestation with giardia. Immunofluorescence of multiple small bowel biopsy specimens showed virtually total absence of IgG, IgM, IgA, and IgD fluorescence, with only slight IgA and IgE staining in the crypt epithelium. Occasionally, in the center of a lymphoid collection, some IgM-positive B-cells could be observed. In order to identify the nature of the lymphoid cells in the nodules, surface membrane characteristics of T- and B-cells were studied by indirect immunoperoxidase histochemistry, using as first layer a highly specific antihuman T-lymphocyte antiserum3.4 respectively, an anti-IgM antiserum, and, as a second layer, peroxidase-labeled antirabbit immunoglobulin serum. Cytoplasmic fluorescence was made visible with antisera to IgG, IgM, IgA, and IgE. of the As can be seen in Figure 1, the vast majority lymphoid cells in the nodules manifested characteristic plasma membrane staining with the anti-T-lymphocyte antiserum, proving that the majority of the accumulating and presumably proliferating lymphocytes were indeed T-lymphocytes. Occasionally, some IgM-positive B-cells were observed, but almost

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Figure 1 o. Nodular lymphoid

cluster, in a suction biopsy specimen from the proximal small intestine, stained with peroxidase-labeled anti-T-lymphocyte antiserum, manifesting the characteristic plasma membrane staining of innumerable T-lymphocytes. b. Negative control in serial section, with, as a first layer, normal rabbit serum; only endogenous peroxidase is active, whereas the lymphoid collection remains unstained.

no IgA, IgM, IgG, and IgE cytoplasmically positive plasma cells could be found. Studies related to the interaction of T- and B-lymphocytes from the peripheral blood during a 6-day stimulation period with pokeweed mitogen” were performed by F. Gmelig-Myeling and A. UytdenHaag, and are summarized in Table 2. The results are expressed as the ratio of the percentage of immunoglobulin containing cells after culture to the initial percentage of B-cells in the medium. Co-cul-

CASE

lune 1979

Table

2.

In Vitro Induction

of Lymphocytic

Cytoplasmic

Ig Staining

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1461

by Pokeweed

Mitogen (6-Day Culture) Number

of cytoplasmic

immunoglobulin-containing

Number

of B-cells put into culture

cells after culture

Ratio

B B B B B

norm. pat. pat. norm. norm.

B pat.

+ + + + + +

T T T T T T

norm. pat. norm. pat. norm. norm.

a B = B-lymphocyte;

214 4 130 5 9 1

+ T pat. + T pat. T = T-lymphocyte;

norm.

= healthy

individual:

pat. = patient.

of normal control B- and T-cells under these conditions led to a large increase of immunoglobulin-containing cells. Such reaction was totally absent when culturing patient B- and T-cells. However, culturing patient B-cells with normal T-cells largely restored the induction of immunoglobulin synthesis. On the other hand, addition of patient T-cells to normal B-cells as well as the addition of patient T-cells to co-cultures of normal B-cells and T-cells did abolish the appearance of cytoplasmic immunoglobulin. These studies strongly suggest that the patient’s Tcell population is primarily responsible for the observed abnormal B-cell function. turing

Follow-up Studies The patient was treated with metronidazole for her giardiasis, with intramuscular administration of gammaglobulin, and, later on, with intravenous infusions of fresh frozen plasma under close supervision of the plasma immunoglobulin levels and for possible appearance of anti-IgA antibodies. The therapy was not very successful, as the patient continued to suffer from general malaise, increasing fatigue, abdominal crampy pain, nausea, anorexia, and more or less constant watery diarrhea, leading to progressive cachexia. Because of the rapid downhill course and the more or less intractable character of the diarrhea, rather resistant to antidiarrheal treatment, a repeated x-ray examination of the small intestine was performed. This showed dramatic extensive destruction, ulceration, and narrowing of the distal small bowel (Figure z), which had been radiologically normal some 8 mo before. Treatment with gammaglobulin infusions and antibiotics was intensified and supplemented with administration of 3-4 liters of plasma/wk, which were necessary to counteract the intestinal protein loss. Because of the rapid deterioration and the impressive destruction of the small bowel, the possibility of a lymphoreticular malignancy was raised. Further investigations, including repeated bone marrow examinations, ab-

Figure

2 a. and b. X-ray examination of the small bowel showing extensive ulcerative destruction and ulceration of the distal small bowel, which was radiologically normal 8 mo before.

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dominal lymphography, and multiple suction biopsies of the diseased ileum, were performed, but they failed to show any evidence of lymphoreticular malignancy. Distal small bowel biopsy specimens showed severe inflammatory changes of the mucosal lining with, in retrospect, evidence of cytomegalovirus infection. A repeat complement fixation test for cytomegalovirus, however, remained negative. Because of the rapid downhill course and because surgical resection was considered impossible in view of the length of small bowel involvement, it was finally decided to treat the patient with corticosteroids in an ultimate attempt to improve the general condition and perhaps to diminish excessive Tsuppressor cell function.” Unfortunately, however, the downhill course continued, and the patient developed massive rectal bleeding and expired. Postmortem examination showed extensive deep serpiginous ulceration and destruction of the ileal mucosa, as illustrated in Figure 3. In addition, there was evidence of severe widespread cytomegalovirus infection not only in the area of ulceration but also in the surrounding mucosa as well as in the esophagus and lung as shown in Figure 3. Detailed electron-

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microscopic findings of the CMV-infected cells are given in Figure 4. There were in addition a few calcified lymph nodes in the mesentery, presumably caused by juvenile bovine tuberculosis, from which the patient had apparently recovered without treatment. There was no evidence of malignancy upon careful extensive search. Postmortem viral cultures from the liver were positive for cytomegalovirus.

Discussion The patient suffered from common variable immunodeficiency. This diagnosis was based upon the clinical history and upon the profound humoral as well as cellular immunodeficiency. The salient features in this patient deserving emphasis relate to the detailed immunologic observations, in particular regarding T- and B-lymphocyte interaction, to the characterization of the lymphocytes in the lymphoid nodules, and to the development of massive ulcerative destruction of the small bowel with evidence of widespread cytomegalovirus infection. The most relevant immunologic disturbances in this patient center around a profound humoral im-

Figure 3 o. Macroscopic view of the extensive destruction and ulceration of the distal small bowel nation. b. and c. Corresponding low- and high-power view of the extensively ulcerated cytomegalic cells with prominent intranuclear inclusion.

mucosa as seen at postmortem examismall bowel. Arrows point to various

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1463

FigIN“e 4 a. Survey electronmicrograph of intranuclear and intracytoplasmic viral inclusions. A portion of the nucleus of an epithelial cell infected with CMV and a rim of surrounding cytoplasm is visible. Numerous CMV-particles can be observed maturing at the periphery of irregular anastomosing strands of electron-dense amorphous material (skeinlike intranuclear inclusions). There is a lack of margination of chromatin. In the cytoplasm, a large cluster of viral particles is visible as coated capsids (X 9,000). b. High-magnification electronmicrograph of viral structures in the nucleus. CMV-particles consist of an inner core (40-50 nm) (short arrow) surrounded by a hexagonal capsid (110 nm) (long arrow) with the individual capsomeres visible at the periphery of the capsid (X 180,000). c. High-magnification of viral structures in the cytoplasm. Enveloped CMV with dense central core, marginated against the inner surface of the capsid, and surrounded by additional lipoprotein envelopes, acquired upon penetration of the nuclear membrane and/or the endoplasmic reticulum (X 180,000).

1464

Figure

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4 d. Characteristic accumulation (short arrow) (X 20,000).

GASTROENTEROLOGY

of abundant

homogeneous

munologic incompetence because of failing B-cell function. Antibody levels to various antigens were indeed unmeasureable. The lamina propria of the gut was largely devoid of immunoglobulin-containing cells, although B-lymphocytes were present in the circulating blood, albeit in small numbers. Recently, Pursz et al.’ described the presence of circulating autoantibodies to B-cells, presumably responsible for the low B-cell number. The presence of such autoantibodies was not determined in our patient. Besides the humoral immune deficit, there was unquestionable disturbance of delayed, cell-mediated immunity, as shown by the abnormal DNCB sensitization and challenge reaction. From a theoretical point of view, several mechanisms may be responsible for the observed abnormal B-cell function such as an intrinsic B-cell defect, an abnormal modulation of B-cell function by either Thelper or T-suppressor cells, an abnormal function of nonlymphoid cells, e.g., monocytes or macrophages, or factor(s) in the serum which modulate the immune response. As shown in this study, the patient’s B-lymphocytes seemed to be able to produce immunoglobulins in vitro, provided they were supplemented with normal T-lymphocytes. For this reason, an intrinsic defect of both B-cells and monocytes appears very unlikely. Moreover, the T-lymphocytes of this patient appear to have a suppressive effect on the capacity of normal control B-cells to synthesize immunoglobulins during prolonged in vitro culture with pokeweed mitogen, a nonspecific B-cell stimulus. Also, Waldmann et al.” and Siegal et al.Y have recently shown that certain patients with

dense

bodies

in the cytoplasm

(long arrow)

mixed

Vol. 76. No. 6

with viral particles

primary or secondary immunodeficiency disorders have increased suppressor-cell activity in their circulating leukocytes. Our studies confirm the observations of the previous authors that co-culture of lymphocytes obtained from normal individuals with the T-lymphocytes from such a patient with immunodeficiency results in marked reduction of the Blymphocyte capacity to synthesize immunoglobulins. Why such patients have abnormal T-suppressor cell activity and to what extent such abnormal Tsuppressor activity explains the profound incompetence of the B-cell system are at present unknown. Moreover, recent studies by Siegal et al.” have shown that such abnormal T-suppressor cell activity can in certain patients be quite variable from time to time. Again, the factors explaining this variability are totally unknown. A further intriguing finding in this disease is the nodular lymphoid hyperplasia, which was radiologically and endoscopically strictly limited to the duodenum in this patient. Until now, the exact nature of the lymphoid elements in these nodules was incompletely understood. Using a highly specific anti-human T-cell antiserum, we were able to show that the vast majority of the lymphocytes in these lymphoid aggregates in the lamina propria and in between the epithelial cells are T-lymphocytes. The trigger for this massive accumulation and proliferation of T-lymphocytes in this condition, however, remains enigmatic. A final and most impressive feature in this patient was the rapid development of very extensive ulcerative destruction of the small bowel, together with

lune 1’379

CASE

evidence of massive cytomegalovirus infection in as well as around the diseased bowel area. Similar ulcerative jejunoileitis as a rare complication of hypogammaglobulinemia has previously been described by Corlin and Pops” as a potentially lethal condition responsible for severe malabsorption and diarrhea. Also, massive cytomegalovirus infection in a patient with common variable immunodeficiency has recently been described by Freeman et al.” In their patient, the most obvious intestinal lesions were mainly localized in the colon. Because of the high number of cytomegalovirus cells at the base of the ulcerative defects, we as well as the previous authors strongly feel that there is a relationship between the overwhelming viral infection and the ulcerative abnormality. Cytomegalovirus infection induces certain unique morphologic changes, illustrate!d in the electron micrographs, such as the reticular appearance of the intranuclear inclusion, the absence of margination of nuclear chromatin, and the presence of characteristic cytoplasmic dense bodies.” Unusual opportunistic infections such as CMV infection should be considered as a cause of diarrhea and intestinal ulceration in patients with immunodeficiency. Serologic tests cannot be used as a diagnostic aid, and one must therefore rely on careful morphologic evaluation and on culture techniques.12

References 1. Eysvoogel tion and Transplant

VP, du Bois R, Melief CJ, et al: Lymphocyte activadestruction in vitro in relation to MLC and HLA. Proc 5:1301-1307, 1973

2.

3.

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Zeylemaker WP, van Oers RH, de Goede RE, et al: Cytotoxic activity of human lymphocytes: Quantitative analysis of Tcell and K-cell cytotoxicity revealing enzyme-like kinetics. J Immunol 119:1507-1514, 1977 Schoorl R, Brute1 de la Riviere A, Von dem Borne AE, et al: Identification of T and B lymphocytes in human breast cancer with immunohistochemical techniques. Am J Path01 84:529-544,

1976

4. Brute1 de la Riviere A, Verhoef-Karssen PR, van Oers MA et al: Preparation and specificity testing of a rabbit anti-human thymocyte serum. VOX Sang 33257-265, 1977 5. Gmelig-Myeling F, UytdenHaag AGC, Ballieux RE: Human Bcell activation in vitro-T-cell-dependent pokeweed mitogeninduced differentiation of blood B lymphocytes. Cell lmmuno1 33:156-169, 1977 6. Saxon A, Stevens RH, Ramer SJ, et al: Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis. J Clin Invest 61:922-930, 1977 7. Tursz T, Preud’Homme J-L, Labaume S, et al: Autoantibodies to B lymphocytes in a patient with hypoimmunoglobulinemia. Characterization and pathogenic role. J Clin Invest 60:405-410, 1977 8. Waldmann TA, Broder S. Krakauer R. et al: The role of suppressor cells in the pathogenesis of common variable hypogammaglobulinaemia and the immunodeficiency associated with multiple myeloma. Fed Proc 35:2067-2072, 1976 9. Siegal FP, Siegal M, Good RA: Suppression of B-cell differentiation by leucocytes from hypogammaglobulinaemia patients. J Clin Invest 58:109-122, 1976 10. Siegal FP, Siegal M, Good RA: Role of helper, suppressor and B-cell defects in the pathogenesis of the hypogammaglobulinemias. N Engl J Med 299:172-178, 1978 11. Corlin RF, Pops MA: Nongranulomatous ulcerative jejunoileitis with hypogammaglobulinemia. Clinical remission after treatment with y-globulin. Gastroenterology 62:473-478, 1972 12. Freeman HI, Shnitka TK, Piercev ” ,IRA. et al: Cvtomenalovirus I _ infection of the gastrointestinal tract in a patient with late onset immunodeficiency syndrome. Gastroenterology 73:13971403, 1977