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Human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa
GASTROENTEROLOGY
1991;100:1521-1527
Human Immunodeficiency Virus Infection of Enterocytes and Mononuclear Cells in Human Jejunal Mucosa CARLA HEISE, SATYA DANDEKAR, PRADEEP KUMAR, RICHARD DUPLANTIER, RICHARD M. DONOVAN, and CHARLES H. HALSTED Department of Internal Medicine, Divisions of Clinical Nutrition Infectious Diseases, University of California, Davis, California
Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function. and weight loss are frequent manifestations of the acquired immunodeficiency syndrome (AIDS) (l-4). Although enteric pathogens such as Giardia lamblia, Cryptosporidium, Mycobacterium avium intracellulare, and cytomegalovirus are often identified in AIDS, chronic diarrhea with intestinal malabsorption has been described in patients infected with the human immunodeficiency virus (HIV) in whom infectious intestinal organisms cannot be identified (l-6). The pathogenesis of diarrhea and intestinal malabsorption in HIV-infected patients has not been determined. Four different groups identified HIV
D
and Metabolism
and
in mononuclear cells in the lamina propria of duodenal and rectal biopsy specimens (5,7-g), although infection of the enterocyte, or intestinal epithelial absorbing cell, has not been reported. The presence of HIV-positive mononuclear cells in the intestinal mucosa of patients with AIDS suggests an immune- or cytokine-mediated secondary mechanism for altered intestinal function (5,lO). The goal of the present study was to evaluate the origin of intestinal malabsorption in HIV-infected patients through detailed study of the structure and function of the intestinal mucosa. Patients were selected at different stages of disease to determine whether intestinal malabsorption precedes the onset of progressive malnutrition in AIDS and to define the intestinal cytopathology of HIV infection before the appearance of other enteric pathogens. Our study provides evidence that both enterocytes and mononuclear cells in the lamina propria are infected by HIV. The data suggest that HIV infection of the intestinal mucosa alters enterocyte maturation and function. As a result, generalized intestinal malabsorption may be an early event and cause of progressive malnutrition in AIDS.
&rhea
Materials
and Methods
Patients Nine
patient volunteers were recruited from the and included one hospitalized and eight ambuhomosexual men, aged 25-57 years, who were sero-
AIDS clinic
latory
Abbreviations used in this paper: AIDS, the acquired immunodeficiency syndrome; ARC, AIDS-related complex; HIV, human immunodeficiency virus. o 1991 by the American Gastroenterological Association
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HEISE ET AL.
low in 2 patients, while plasma zinc, iron, and magnesium were each depressed in one third of the group (Table 1).
positive for HIV by Western blot assay and had experienced recurrent diarrhea for 2-8 weeks before evaluation. Three patients gave a history of IV drug use. Five patients were diagnosed with AIDS on the basis of previous or concurrent (one patient) Pneumocystis carinii pneumonia, and the remaining four patients had AIDS-related complex (ARC) (11). One patient was receiving oral zidovudine, two patients were receiving oral antifungal agents, and one patient was receiving IV pentamidine in the hospital. The T-lymphocyte helper-suppressor ratio was reversed in eight of the nine patients. Three patients with AIDS had absolute T4-lymphocyte counts < 200/mm3. Four of the nine patients were positive for serum p24 antigen (HTLV III Antigen EAI; Abbott Laboratories, Chicago, IL). Enteric infections diagnosed by intestinal biopsy included G. lamblia in one patient with ARC and M. avium intracellulare in one patient with AIDS. Stool microbiology was negative for other Cryptosporidia), and Microsporidia pathogens (including was excluded by electron microscopy of jejunal biopsies. Ten healthy patients with functional bowel syndrome and normal intestinal absorption and biopsies served as normal controls for morphometry and in situ hybridization studies. Normal values for enzyme activities have been established in our laboratory using jejunal biopsy samples normal values (12) were from 26 normal subjects. Published used for the intestinal absorption studies. Biopsy samples from 2 adult patients with untreated celiac sprue were included as disease controls for cytospectrophotometry. The protocol was approved by the Human Subjects Review Committee of the University of California, Davis, and informed consent was obtained from all patient volunteers.
Nutritional
Intestinal
Jejunal
Morphometry Eyepiece micrometer measurements were made in blinded fashion of villus height and crypt depth using 5-10 well-oriented villi from each specimen. Enterocyte nuclear size and density were measured by cytospectrophotometry,
Table I. Clinical Laboratory Data for Human Immunodeficiency
Mean + SD Range of values No. of values below normal range ARC AIDS
Red blood cell folate
WI (35-50)
WI (140-165)
(nmoliL] (>450)
30 + 38 24-50
114 2 21 77-132
013
214
Biopsy Specimens
Peroral jejunal biopsy specimens were obtained by the Quinton hydraulic multiple biopsy apparatus (Quinton Instrument Co., Seattle, WA) or by upper gastrointestinal endoscopy. Specimens were fixed in Bouin’s solution, embedded in paraffin, and stained with H&E for light microscopy studies. Specimens were fixed in Karnofsky’s solution for electron microscopy. Other specimens were immediately covered with OCT Compound (Miles Scientific, Naperville, IL), frozen in a dry ice and acetone bath, and stored at -70°C. Frozen specimens were used for subsequent activity measurements of sucrase and lactase (18). Mucosal biopsies were also used to measure brush border folate hydrolase, an enzyme required for the digestion of dietary folates before intestinal transport (19).
No differences were observed in anthropometric measurements between patients with ARC and AIDS. Three patients had no weight loss, and the remainder averaged 7% loss of body weight during the 6 months preceding evaluation. Triceps skinfold thickness, 24% to 93% of standard, indicated reduction of body fat in the majority of patients, whereas midarm muscle circumference was 84% to 113% of standards for men, indicating preservation of lean body mass. All patients were mildly anemic, and 2 had serum albumin levels below the normal range. The red blood cell folate was below normal in 1 patient, and plasma folate was
Hemoglobin
Absorption Studies
Patients were instructed to follow diets containing 100 g of fat per day for 6 days and to collect a fecal pool on the last 3 days. Daily fat intakes were estimated by computerized nutrient analysis and ranged from 88 to 152 g. Fecal fat excretion was measured after lipid extraction of the 3-day stool collection (15) and is expressed as a percentage of estimated daily fat intake recovered in the stool per day. Other tests performed in fasting patients included D-xylose absorption by measurement of serum levels at 2 hours, s-hour urinary excretion following administration of a 26-g oral dose (16), and the absorption of a standard 50-g oral dose of lactose by analysis of hydrogen in breath samples (17) obtained at 15minute intervals for 1 hour and at 39minute intervals for 2 hours using a Quintron Model 12 microlyzer (Quintron Instrument Co., Inc., Milwaukee, WI).
Assessment
Serum albumin
Vol. 100, No. 6
Virus-Infected
Plasma folate (nmoZ/L)
Patients
Plasma vitamin B,,
Plasma zinc
Plasma iron
Plasma magnesium
C>ll)
@moZ/L) (150-660)
(pmol/L) (11.5-18.5)
bmol/L) (9-27)
(0.80-1.20)
1080 + 375 320-1680
35 * 23 4-78
310 k 180 160-730
12.4 + 2.3 9.0-15.3
17 2 10 5-38
0.78 + 0.06 0.66-0.86
313
O/4
o/4
O/4
l/4
l/4
114
414
l/5
215
o/5
214
215
315
(mmol/L)
NOTES. Normal values are given in parentheses (12,13). Plasma and red blood cell folate levels were measured by the microbiological assay with Luctobacillus casei (141,vitamin B,, by radioassay (Becton-Dickinson, New York, NY], and plasma zinc, iron, and magnesium by atomic absorption spectrophotometry (Perkin Elmer Model 3030B)
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HIV INFECTION
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1523
using a microscope (Zeiss, Inc., Thornwood, NY), an attached charge coupled device camera (CCD Model 4800; Cohu Instruments, San Diego, CA), and a data translation video analog to digital converter interfaced with a Micro VAX-II computer (Digital Equipment Co., Maynard, CA) (20). The computer program segmented the microscopic images and calculated area and total optical density. Statistical differences between control biopsies and HIVinfected groups were determined by repeated measures analysis of variance.
In Situ Hybridization To detect posttranscriptional RNA (tRNA) and viral replication in HIV-infected cells, we performed in situ hybridization with an “‘S-labeled DNA probe using a modification of the procedure of Brahic and Haase (21). Tissue sections of paraffin-embedded jejunal biopsies were deparaffinized and hybridized with ‘“S-labeled DNA probe (9Kb pBHl0 fragment of HIV-l, obtained from R. Gallo, National Cancer Institute) to a specific activity of 1 X lOa cpm/pg or greater. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 50 mmol/L NaH,PO,, 0.6 mol/LNaCl, 0.5 mmol/Lethylenediaminetetraacetic acid (EDTA), 1 x Denhardt’s solution, 75 &mL Escherichia coli tRNA, 100 &mL salmon sperm DNA, and 20 mmoI/‘L dithiothreitol. The slides were washed, dehydrated, and autoradiographed with NTB2 emulsion (International Biotechnologies Inc., New Haven, CT) for 3-14 days, developed with Kodak D-19 (Eastman Kodak Co, Rochester, NY), and counterstained with hematoxylin. Tissue sections were also hybridized after treatment with RNAse to verify that positive signals were specific for HIV-RNA (21).
Results
Figure 1. Brush horder disaccharidase activities and sucrasei lactase ratios of jejunal biopsy samples from HIV-infected patients. Enzyme activities are expressed as units per milligram protein; 1 mIJ is equal to 1 nmol of substrate hydrolyzed per minute. The normal ranges are shown by the shaded bars and are based on measurements from 26 normal biopsy specimens. ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. avium intracellular-e.
but was not significantly different between patient and control groups. Lactose intolerance was found in 4 patients, including 3 with lactase levels < 10 mU per mg protein (Table 2). Elevated baseline breath H, was found in 1 patient, suggestive of bacterial stasis. Intestinal brush border folate hydrolase activity was below the normal range in 4 patients, and a subnormal red cell folate level was found in 1 patient. Among the HIV-infected patients, the activity of brush border folate hydrolase was positively correlated with the red cell folate level (I. = 0.75, P < 0.05) (Figure 2).
Intestinal Function Steatorrhea was found in 5 of the 6 patients who completed the 72-hour fecal collection (Table 2). Two patients excreted > 10% and 1 patient excreted > 20% of dietary fat intake. Three patients demonstrated D-xylose malabsorption by low levels in both blood and urine. Compared with our normal values, the activity of mucosal sucrase was low in 6 patients, and the activity of lactase was decreased in all 9 HIV-infected patients (Figure 1). The ratio of sucrase activity to lactase activity was elevated in 2 patients
Jejunal Morphology Jejunal biopsy specimens from HIV-infected patients were interpreted as normal in 2 specimens, whereas 5 specimens showed nonspecific inflammatory changes and 2 specimens included enteric pathogens (G. lamblia and M. avium intracellulare). Electron microscopy detected no organisms, and intranuclear inclusion bodies characteristic of cytomegalovirus were absent. Biopsy specimens from control patients were all pathologically normal, whereas both
Table 2. intestinal Absorption o-Xylose
Fat Fecal (% dietaryintoke) Normal values Patients abnormal ARC AIDS Range of abnormal values
<6 414 l/2 7-23
Lactose Blood level (mg/dL at 2 h)
Maximum increase of breath H,
(g/s hl > 4.5
>30
<20
o/4 313 0.4-2.8
o/4 313 17-28
214 215 33-55
Urine excretion
@pm K)
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GASTROENTEROLOGY
HEISE ET AL.
0
10
Brush
Border mwmg
20
Folato
30
Hydrolaw
potcin
Figure 2. Correlation between red cell folate levels and jejunal brush border folate hydrolase activity in HIV-infected patients. The normal value for brush border folate hydrolase is 18-45 mIJ/mg protein (19) and > 450 nmol/L for red cell folate (13). ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. aviam iatracellulare.
celiac sprue biopsy specimens showed typical flattened villus morphology. Villus heights in biopsy specimens from all HIV-infected patients were within 2SD of our normal mean of 400 pm, although values from 5 biopsy specimens were < 300 km (Figure 3). Crypt depth was greater than the normal mean of 150 Frn in 7 patients and was greater than the mean + 2SD in 3 patients. The villus height to crypt depth ratio was less than the normal mean of 2.7:l.O in all patients and less than the mean -2SD in 7 patients. The percentage of mitotic figures was greater than the normal mean of 2.0% in 8 patients and greater than the upper range of control in 3 patients (Figure 3).
Compared with findings in biopsy specimens from normal controls, cytospectrophotometric analysis of the villus tips showed significantly enlarged enterocyte nuclei in 8 and a decreased number of nuclei in 6 of the 9 HIV-infected patients (Figure 4). The mean enterocyte nuclear size in biopsy samples from HIV patients was approximately 1.5 times larger than the enterocyte nuclear size in the control biopsy samples (P < 0.001). The density of enterocyte nuclei was lower in HIV-infected biopsy specimens than in control biopsy specimens (P < 0.001). Mean values for both measurements in celiac biopsy specimens were significantly different from controls (P < 0.05) but similar to the HIV-infected patients. No significant differences in cytospectrophotometric measurements were seen between patients with ARC and AIDS.
Human immunodeficiency virus RNA was detected by in situ hybridization in 5 of 9 patient jejunal biopsy specimens. All 5 HIV-infected biopsy samples showed evidence of chronic inflammation. Human immunodeficiency virus infection was identified in 4-6 enterocytes per section in villi of one patient who had normal circulating T-helper cells but abnormal cytospectrophotometry, steatorrhea, and lactose intolerance (Figure 5). Most enterocytes did not support
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VilluslCrypt
Mitotic
Index
i
Figure 3. Villus height, crypt depth, villus length to crypt depth ratios, and mitotic indices of jejunal biopsy specimens from HIV-infected patients. Patient values for villus and crypt measurements are means of 3-10 well-oriented villi per biopsy sample. The normal ranges (shaded bars) represent means ?2SD of 93 measurements from 10 normal specimens. The mitotic index is the number of mitotic figures in a minimum of 300 crypt epithelial cells per biopsy specimen and is expressed as a percentage for each biopsy specimen. The normal range for mitotic index is based on measurements from 10 normal biopsy specimens. ?? , ARC; El, ARC + G. lamblia; 0, AIDS: 0, AIDS + M. aviam iatracellulare.
5 F 9
:: ................ 3 0
9 5 52. c
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Nuclear
Size
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Crypt
.
. 2.0
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0 height
4.0 9-
Vol. 100, No. 6
II
. 00
1
Nuclear
4
0-l density
Figure 4. Cytospectrophotometric measurements of enterocyte nuclear size and density (number of nuclei per 100 km) in jejunal biopsy specimens from HIV-infected and celiac sprue patients and control subjects. Individual values are the mean of 100 measurements from each biopsy sample, using enterocytes located in the top third of the villus. Significant differences were found between HIV-infected and control biopsy samples for both enterocyte nuclear size (34 k 0.3 vs. 22 2 0.2 urn’; P < 0.001) and enterocyte nuclear density (20 k 1.7 vs. 30 k 3.2 nuclei/100 pm; P < 0.001). Mean values in celiac sprue biopsy specimens for both nuclear size (37 2 5.6) and density (18 f 2.5) were significantly different from controls (P < 0.05) but not different from HIV-infected patients. Shaded bars indicate normal ranges. ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. avium intracellulare.
June 1991
HIV INFECTION
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Figure 5. The presence of HIV RNA detected by in situ hyhridization in villus enterocytes of a patient with ARC. The patient had been diagnosed as seropositive for HIV 1 month before our evaluation and had a Z-month history of diarrhea. The CD4-CD8 ratio was normal (1.5) with 426 T-helper cells/mn?. Severe fat malabsorption (23% of dietary intake) and lactose intolerance (55 ppm H, over baseline) with negligible lactase activity were found. Villus length to crypt depth ratio (2.l:l.O) and mitotic index (1.52%) were both within our normal ranges, whereas the mean enterocyte nuclear size was above normal at 33 pm’. A.
Mid infected
villus of jejunal biopsy specimen enterocytes (original magnification
B. Higher magnification intensity of HIV RNA X1.250: bar = 10 wm).
showing two HIVx500; bar = 20 p,m).
of Figure 5A demonstrating high-signal in enterocytes (original magnification
C. Different field from same biopsy specimen showing another infected enterocyte with adjacent intraepithelial mononuclear cell (original magnification X1250; bar = 10 km).
viral replication to the same degree as the mononuclear cells, as evidenced by the need for a longer exposure time for detection (2 weeks vs. 5 days) and by reduced density of silver grains within the cells. Schmorl’s stain for argentaffin granules (22) identified enterochromaffin cells in serial sections, all separate from HIV-infected cells. The remaining four positive biopsy specimens contained HIV-infected mononuclear cells in the intraepithelial spaces, the subepithelium, the lamina propria, and near the crypt base (Figure 6). Jejunal biopsy specimens from uninfected control subjects were negative for HIV RNA. Positive and negative signals were obtained from HIV-infected and uninfected cultured T cells (CEM line). No positive cells were detected following RNAse treatment of tissue, with hybridization with pSP64 vector alone or with a probe to the simian immunodeficiency virus, indicating that positive signals were specific for HIV RNA.
Discussion The present study confirms that intestinal malabsorption can occur in an early stage and may be an important cause of malnutrition in AIDS. Jejunal mucosal HIV infection in our patients was associated with altered enterocyte differentiation and function, as shown by reversal of the villus length to crypt depth ratio, increased enterocyte nuclear size, and depression of digestive enzyme activity. The absence of other identifiable intestinal pathogens suggests a direct role for HIV in the pathogenesis of AIDS enteropathy and the potential for antiviral therapy directed at the mucosal lesion. Others have demonstrated HIV infection in the intestine by in situ hybridization (7-9) and immunohistochemical (5) techniques. Infected enterochromaffin cells, lymphocytes, and macrophages have been identified in rectal biopsy specimens (7,9,23),
1526
HEISE ET AL.
Figure 6. HIV RNA in two subepitbelial mononuclear cells in the jejunal mucosa of a patient with AIDS (original magnification X 1250; bar = 10 pm).
and two studies (7,8) suggest that rectal crypt epithelial cells are capable of infection with HIV. In jejunal biopsy specimens, HIV antigen was found previously in lamina propria mononuclear cells and in occasional intraepithelial lymphocytes (5). Our data provide the first evidence of jejunal enterocyte infection by HIV. The finding of direct enterocyte infection with HIV in one biopsy specimen and lamina propria mononuclear cell infection in four others suggests a wider presence of undetected HIV in the jejunal mucosa. The method of in situ hybridization for RNA identifies cells that are actively expressing viral nucleic acid but does not detect cells in a latent stage of infection. Although it is not known whether the CD4 receptor is present on enterocytes, the CD4 receptor RNA was expressed in the HT29 colon carcinoma cell line and in normal colon cells (24). Two colon carcinoma cell lines (HT29 and SW480) supported productive HIV infection in vitro (24), and HIV was shown to replicate in normal and malignant intestinal cell cultures (25). In another tissue, renal tubule and glomerular epithelial cells were found capable of infection by HIV in vivo (26). The route of jejunal mucosal infection by circulating blood or direct contact with HIV in the intestinal lumen cannot be determined by our studies. Previous studies reported nonspecific inflammation, partial villus atrophy (1,5), and increased number of intraepithelial lymphocytes (1,27) in the intestinal mucosa of jejunal biopsy samples from patients with AIDS. A study of mucosal abnormalities in HIV-infected patients documented increased crypt depth and decreased mitoses together with decreased
GASTROENTEROLOGY
Vol. 100, No. 6
lactase activity (5). Another study of jejunal biopsy specimens in ambulatory HIV-infected patients described increased jejunal villus to crypt ratio with increased mitoses (28). In our study of early infection, the villus length to crypt depth ratio was reversed equally in AIDS and ARC biopsy samples, and increased numbers of mitoses were found in three of nine patients. Among the studies, the difference in observed mitotic indices may relate to duration and severity of the disease, so that mitoses are more likely to be decreased in hospitalized patients at later stages of AIDS. Altered cell proliferation could result in functionally immature enterocytes with enlarged nuclei, as has been described in the mucosal disease tropical sprue (29). The finding by cytospectrophotometry that enterocyte nuclear size was increased and the numbers of enterocytes per unit villus length were decreased equally in both ARC and AIDS (Figure 4) suggests that immature enterocytes with larger nuclei are produced at early stages of infection. Enterocyte immaturity could account for the decreased expression of brush border enzymes in all our patients and for generalized malabsorption of both dietary fat and orally administered n-xylose observed in about half the patients. The finding of steatorrhea in all patients with ARC and the most severe fat malabsorption in a patient with HIV-infected enterocytes underscores the relationships among mucosal HIV infection, enterocyte immaturity, and dysfunction. Finding a uniform incidence of low lactase levels in the HIV-infected patients is not surprising because this enzyme is less abundant than sucrase in the intestinal mucosa and hence more susceptible to significant inhibition. Nevertheless, finding a similar ratio of sucrase to lactase in HIV-infected patients and controls suggests nonselective suppression of brush border enzyme activity. Finding low jejunal lactase in all patients, but lactose intolerance by the breath hydrogen test in only 4 patients, suggests that the enzymatic defect precedes its clinical manifestation. Finding levels of brush border folate hydrolase below our published norm (19) in half of the patients (Figure 2) is consistent with the generalized effect of HIV infection on brush border hydrolases. The correlation of low brush border folate hydrolase with red cell folate levels (Figure 2) suggests a regulatory effect of this enzyme on the supply of folate to tissues. Although evidence for HIV infection of the jejunal mucosa is now quite convincing, the pathogenesis of the observed mucosal change is speculative. Possible explanations include retarded cell maturation due to low mucosal folate, a direct effect of HIV on enterocyte function, or an indirect cytokine-mediated effect on cell proliferation and maturation.
June
1991
HIV INFECTION
References 1. Kotler
DP, Gaetz HP, Lange M, Klein EB, Holt PR. Enteropathy associated with the acquired immunodeficiency syndrome. AnnInternMed 1984;101:421-428.
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2. Dworkin B, Wormser GP, Rosenthal WS, Heier SK, Braunstein M, Weiss L, Jankowski R, Levy D, Weiselberg S. Gastrointestinal
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3. Gillin JS, Shike M, Alcock N, Urmacher C, Krown S, Kurtz RC, Lightdale CJ, Winawer SJ. Malabsorption and mucosal abnormalities of the small intestine in the acquired immunodeficiency syndrome. Ann Intern Med 1985;?02:619-622. 4. Smith PD, Lane HC, Gill VJ, Manischewitz JF, Quinnan GV, Fauci AS, Masur H. Intestinal infections in patients with the acquired immunodeficiency syndrome (AIDS). Ann Intern Med 1988;108:328-333. 5. Ullrich R, Zeitz M, Heise W, L’age M, Hoffken G, Riecken EO. Small intestinal structure and function in patients infected with human immunodeficiency virus (HIV): evidence for HIVinduced enteropathy. Ann Intern Med 1989;111:15-21. 6. Miller ARO, Griffin GE, Batman P, Farquar C, Forster SM. Pinching AJ, Harris JRW. Jejunal mucosal architecture and fat absorption in male homosexuals infected with human immunodeficiency virus. Q J Med 1988;69:1009-1019. 7. Nelson JA, Wiley CA, Reynolds-Kohler C, Reese CE, Margaretten W, Levy JA. Human immunodeficiency virus detected in bowel epithelium from patients with gastrointestinal symptoms. Lancet i988;1:259-262. a. Mathijs JM, Hing M, Grierson J, Dwyer DE, Godschmidt C, Cooper DA, Cunningham AL. HIV infection of rectal mucosa (letter). Lancet i9aa;i:iiii. 9. Fox CH, Kotler D, Tierney A, Wilson CS, Fauci AS. Detection of HIV-1 RNA in the lamina propria of patients with AIDS and gastrointestinal disease. J Infect Dis 1989;159:467-471. 10. MacDonald TT, Spencer J. Evidence that activated mucosal T cells play a role in the pathogenesis of enteropathy in human small intestine. J Exp Med 1988;167:1341-1349. 11. Leads from the MMWR. Revision of the CDC Surveillance case definition for acquired immunodeficiency syndrome. JAMA 1987;258:1143-1154. 12. Halsted JA, Halsted CH, eds. The laboratory in clinical medicine: interpretation and application. Philadelphia: Saunders, 1981:1027-1028. 13. Herbert V, Colman N. Folic acid and vitamin B12. In: Shils ME, Young VR, eds. Modern nutrition in health and disease. 7th ed. Philadelphia: Lea & Fe’oiger, 1988:388-416. 14. Tamura T, Shin YS, Williams MA, Stokstad ELR. Lactobacillus casei response to pteroylpolyglutamates. Anal Biochem 1972; 49:517-521. 15. Van de Kamer JH. Total fatty acids in stool. In: Seligson D, ed.
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Standard methods of clinical chemistry. Volume II. New York: Academic, 1958:34-39. Roe JH, Rice EW. A photometric method for determination of free pentoses in animal tissues. JBiol Chem 1948;173:507-512. Newcomber AD, McGill DB. Thomas PJ, Hoffman AF. Prospective comparison of indirect methods for detecting lactase deficiency. N Engl J Med 1975;293:1232-1235. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biothem 1968;22:99-107. Halsted CH, Beer WH, Chandler CJ, Ross K. Wolfe BM, Bailey L, Cerda JJ. Clinical studies of intestinal folate conjugases. J Lab Clin Med 1986;107:228-232. Goldstein E, Donovan RM, Kim Y. Applications of computerized microscopic image analysis in infectious diseases. Rev Infect Dis 1988;10:92-102. Brahic M, Haase AT. Detection of viral sequences of low reiteration frequency by jn situ hybridization. Proc Nat1 Acad Sci 1978;75:6125-6129. Perls M. Nachweis von Eisenoxyd in Oewissen Pigmenten. Virchows Arch Path01 Anat 1867;39:42-48. Levy JA, Margaretten W, Nelson J. Detection of HIV in enterochromaffin cells in the rectal mucosa of an AIDS patient. Am J 1989;84:787-789. Gastroenterol Adachi A, Koenig S, Gendelman HE, Daugherty D, Gatton-Celli S, Fauci AS, Martin MA. Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus. J Virol 1987;61:209-213. Moyer MP, Ramirez A, McKee-Suarez MS, Gendelman HI. In: Sixth International Conference on AIDS. HIV infection of gastrointestinal (GI) cell cultures (abstr). Volume II. San Francisco: 1990:140. Cohen AH, Sun NCJ, Shapshak P, Imagawa DT. Demonstration of human immunodeficiency virus in renal epithelium in HIV-associated nephropathy. Mod Path01 1989:2:125-128. Weber JR, Dobbins WO. The intestinal and rectal epithelial lymphocyte in AIDS. Am J Surg Pathol 1986;10:627-639. Cummins AG, LaBrooy JT, Stanley DP, Rowland R, Shearman DJC. Quantitative histological study of enteropathy associated with HIV infection. Gut 1990;31:317-321. Swanson VL, Thomassen RW. Pathology of the jejunal mucosa in tropical sprue. Am J Path01 1965;46:511-551.
Received April 11, 1990. Accepted November 2, 1990. Address requests for reprints to: Charles H. Halsted, M.D., Division of Clinical Nutrition and Metabolism, TB 156, University of California, Davis, California 95616. The authors acknowledge the technical expertise of David Vinson, Carol Chandler, Rich Peterson, Dale J. Martfeld, and Robert Munn. Supported by grants from the University of California AIDS Task Force; from the National Institutes of Health, DK-35747 and R29-NS27338; and from the Bristol-Myers Squibb Company.
1991;100:1521-1527
Human Immunodeficiency Virus Infection of Enterocytes and Mononuclear Cells in Human Jejunal Mucosa CARLA HEISE, SATYA DANDEKAR, PRADEEP KUMAR, RICHARD DUPLANTIER, RICHARD M. DONOVAN, and CHARLES H. HALSTED Department of Internal Medicine, Divisions of Clinical Nutrition Infectious Diseases, University of California, Davis, California
Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function. and weight loss are frequent manifestations of the acquired immunodeficiency syndrome (AIDS) (l-4). Although enteric pathogens such as Giardia lamblia, Cryptosporidium, Mycobacterium avium intracellulare, and cytomegalovirus are often identified in AIDS, chronic diarrhea with intestinal malabsorption has been described in patients infected with the human immunodeficiency virus (HIV) in whom infectious intestinal organisms cannot be identified (l-6). The pathogenesis of diarrhea and intestinal malabsorption in HIV-infected patients has not been determined. Four different groups identified HIV
D
and Metabolism
and
in mononuclear cells in the lamina propria of duodenal and rectal biopsy specimens (5,7-g), although infection of the enterocyte, or intestinal epithelial absorbing cell, has not been reported. The presence of HIV-positive mononuclear cells in the intestinal mucosa of patients with AIDS suggests an immune- or cytokine-mediated secondary mechanism for altered intestinal function (5,lO). The goal of the present study was to evaluate the origin of intestinal malabsorption in HIV-infected patients through detailed study of the structure and function of the intestinal mucosa. Patients were selected at different stages of disease to determine whether intestinal malabsorption precedes the onset of progressive malnutrition in AIDS and to define the intestinal cytopathology of HIV infection before the appearance of other enteric pathogens. Our study provides evidence that both enterocytes and mononuclear cells in the lamina propria are infected by HIV. The data suggest that HIV infection of the intestinal mucosa alters enterocyte maturation and function. As a result, generalized intestinal malabsorption may be an early event and cause of progressive malnutrition in AIDS.
&rhea
Materials
and Methods
Patients Nine
patient volunteers were recruited from the and included one hospitalized and eight ambuhomosexual men, aged 25-57 years, who were sero-
AIDS clinic
latory
Abbreviations used in this paper: AIDS, the acquired immunodeficiency syndrome; ARC, AIDS-related complex; HIV, human immunodeficiency virus. o 1991 by the American Gastroenterological Association
0016-5085/91/.$3.00
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GASTROENTEROLOGY
HEISE ET AL.
low in 2 patients, while plasma zinc, iron, and magnesium were each depressed in one third of the group (Table 1).
positive for HIV by Western blot assay and had experienced recurrent diarrhea for 2-8 weeks before evaluation. Three patients gave a history of IV drug use. Five patients were diagnosed with AIDS on the basis of previous or concurrent (one patient) Pneumocystis carinii pneumonia, and the remaining four patients had AIDS-related complex (ARC) (11). One patient was receiving oral zidovudine, two patients were receiving oral antifungal agents, and one patient was receiving IV pentamidine in the hospital. The T-lymphocyte helper-suppressor ratio was reversed in eight of the nine patients. Three patients with AIDS had absolute T4-lymphocyte counts < 200/mm3. Four of the nine patients were positive for serum p24 antigen (HTLV III Antigen EAI; Abbott Laboratories, Chicago, IL). Enteric infections diagnosed by intestinal biopsy included G. lamblia in one patient with ARC and M. avium intracellulare in one patient with AIDS. Stool microbiology was negative for other Cryptosporidia), and Microsporidia pathogens (including was excluded by electron microscopy of jejunal biopsies. Ten healthy patients with functional bowel syndrome and normal intestinal absorption and biopsies served as normal controls for morphometry and in situ hybridization studies. Normal values for enzyme activities have been established in our laboratory using jejunal biopsy samples normal values (12) were from 26 normal subjects. Published used for the intestinal absorption studies. Biopsy samples from 2 adult patients with untreated celiac sprue were included as disease controls for cytospectrophotometry. The protocol was approved by the Human Subjects Review Committee of the University of California, Davis, and informed consent was obtained from all patient volunteers.
Nutritional
Intestinal
Jejunal
Morphometry Eyepiece micrometer measurements were made in blinded fashion of villus height and crypt depth using 5-10 well-oriented villi from each specimen. Enterocyte nuclear size and density were measured by cytospectrophotometry,
Table I. Clinical Laboratory Data for Human Immunodeficiency
Mean + SD Range of values No. of values below normal range ARC AIDS
Red blood cell folate
WI (35-50)
WI (140-165)
(nmoliL] (>450)
30 + 38 24-50
114 2 21 77-132
013
214
Biopsy Specimens
Peroral jejunal biopsy specimens were obtained by the Quinton hydraulic multiple biopsy apparatus (Quinton Instrument Co., Seattle, WA) or by upper gastrointestinal endoscopy. Specimens were fixed in Bouin’s solution, embedded in paraffin, and stained with H&E for light microscopy studies. Specimens were fixed in Karnofsky’s solution for electron microscopy. Other specimens were immediately covered with OCT Compound (Miles Scientific, Naperville, IL), frozen in a dry ice and acetone bath, and stored at -70°C. Frozen specimens were used for subsequent activity measurements of sucrase and lactase (18). Mucosal biopsies were also used to measure brush border folate hydrolase, an enzyme required for the digestion of dietary folates before intestinal transport (19).
No differences were observed in anthropometric measurements between patients with ARC and AIDS. Three patients had no weight loss, and the remainder averaged 7% loss of body weight during the 6 months preceding evaluation. Triceps skinfold thickness, 24% to 93% of standard, indicated reduction of body fat in the majority of patients, whereas midarm muscle circumference was 84% to 113% of standards for men, indicating preservation of lean body mass. All patients were mildly anemic, and 2 had serum albumin levels below the normal range. The red blood cell folate was below normal in 1 patient, and plasma folate was
Hemoglobin
Absorption Studies
Patients were instructed to follow diets containing 100 g of fat per day for 6 days and to collect a fecal pool on the last 3 days. Daily fat intakes were estimated by computerized nutrient analysis and ranged from 88 to 152 g. Fecal fat excretion was measured after lipid extraction of the 3-day stool collection (15) and is expressed as a percentage of estimated daily fat intake recovered in the stool per day. Other tests performed in fasting patients included D-xylose absorption by measurement of serum levels at 2 hours, s-hour urinary excretion following administration of a 26-g oral dose (16), and the absorption of a standard 50-g oral dose of lactose by analysis of hydrogen in breath samples (17) obtained at 15minute intervals for 1 hour and at 39minute intervals for 2 hours using a Quintron Model 12 microlyzer (Quintron Instrument Co., Inc., Milwaukee, WI).
Assessment
Serum albumin
Vol. 100, No. 6
Virus-Infected
Plasma folate (nmoZ/L)
Patients
Plasma vitamin B,,
Plasma zinc
Plasma iron
Plasma magnesium
C>ll)
@moZ/L) (150-660)
(pmol/L) (11.5-18.5)
bmol/L) (9-27)
(0.80-1.20)
1080 + 375 320-1680
35 * 23 4-78
310 k 180 160-730
12.4 + 2.3 9.0-15.3
17 2 10 5-38
0.78 + 0.06 0.66-0.86
313
O/4
o/4
O/4
l/4
l/4
114
414
l/5
215
o/5
214
215
315
(mmol/L)
NOTES. Normal values are given in parentheses (12,13). Plasma and red blood cell folate levels were measured by the microbiological assay with Luctobacillus casei (141,vitamin B,, by radioassay (Becton-Dickinson, New York, NY], and plasma zinc, iron, and magnesium by atomic absorption spectrophotometry (Perkin Elmer Model 3030B)
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HIV INFECTION
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using a microscope (Zeiss, Inc., Thornwood, NY), an attached charge coupled device camera (CCD Model 4800; Cohu Instruments, San Diego, CA), and a data translation video analog to digital converter interfaced with a Micro VAX-II computer (Digital Equipment Co., Maynard, CA) (20). The computer program segmented the microscopic images and calculated area and total optical density. Statistical differences between control biopsies and HIVinfected groups were determined by repeated measures analysis of variance.
In Situ Hybridization To detect posttranscriptional RNA (tRNA) and viral replication in HIV-infected cells, we performed in situ hybridization with an “‘S-labeled DNA probe using a modification of the procedure of Brahic and Haase (21). Tissue sections of paraffin-embedded jejunal biopsies were deparaffinized and hybridized with ‘“S-labeled DNA probe (9Kb pBHl0 fragment of HIV-l, obtained from R. Gallo, National Cancer Institute) to a specific activity of 1 X lOa cpm/pg or greater. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 50 mmol/L NaH,PO,, 0.6 mol/LNaCl, 0.5 mmol/Lethylenediaminetetraacetic acid (EDTA), 1 x Denhardt’s solution, 75 &mL Escherichia coli tRNA, 100 &mL salmon sperm DNA, and 20 mmoI/‘L dithiothreitol. The slides were washed, dehydrated, and autoradiographed with NTB2 emulsion (International Biotechnologies Inc., New Haven, CT) for 3-14 days, developed with Kodak D-19 (Eastman Kodak Co, Rochester, NY), and counterstained with hematoxylin. Tissue sections were also hybridized after treatment with RNAse to verify that positive signals were specific for HIV-RNA (21).
Results
Figure 1. Brush horder disaccharidase activities and sucrasei lactase ratios of jejunal biopsy samples from HIV-infected patients. Enzyme activities are expressed as units per milligram protein; 1 mIJ is equal to 1 nmol of substrate hydrolyzed per minute. The normal ranges are shown by the shaded bars and are based on measurements from 26 normal biopsy specimens. ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. avium intracellular-e.
but was not significantly different between patient and control groups. Lactose intolerance was found in 4 patients, including 3 with lactase levels < 10 mU per mg protein (Table 2). Elevated baseline breath H, was found in 1 patient, suggestive of bacterial stasis. Intestinal brush border folate hydrolase activity was below the normal range in 4 patients, and a subnormal red cell folate level was found in 1 patient. Among the HIV-infected patients, the activity of brush border folate hydrolase was positively correlated with the red cell folate level (I. = 0.75, P < 0.05) (Figure 2).
Intestinal Function Steatorrhea was found in 5 of the 6 patients who completed the 72-hour fecal collection (Table 2). Two patients excreted > 10% and 1 patient excreted > 20% of dietary fat intake. Three patients demonstrated D-xylose malabsorption by low levels in both blood and urine. Compared with our normal values, the activity of mucosal sucrase was low in 6 patients, and the activity of lactase was decreased in all 9 HIV-infected patients (Figure 1). The ratio of sucrase activity to lactase activity was elevated in 2 patients
Jejunal Morphology Jejunal biopsy specimens from HIV-infected patients were interpreted as normal in 2 specimens, whereas 5 specimens showed nonspecific inflammatory changes and 2 specimens included enteric pathogens (G. lamblia and M. avium intracellulare). Electron microscopy detected no organisms, and intranuclear inclusion bodies characteristic of cytomegalovirus were absent. Biopsy specimens from control patients were all pathologically normal, whereas both
Table 2. intestinal Absorption o-Xylose
Fat Fecal (% dietaryintoke) Normal values Patients abnormal ARC AIDS Range of abnormal values
<6 414 l/2 7-23
Lactose Blood level (mg/dL at 2 h)
Maximum increase of breath H,
(g/s hl > 4.5
>30
<20
o/4 313 0.4-2.8
o/4 313 17-28
214 215 33-55
Urine excretion
@pm K)
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GASTROENTEROLOGY
HEISE ET AL.
0
10
Brush
Border mwmg
20
Folato
30
Hydrolaw
potcin
Figure 2. Correlation between red cell folate levels and jejunal brush border folate hydrolase activity in HIV-infected patients. The normal value for brush border folate hydrolase is 18-45 mIJ/mg protein (19) and > 450 nmol/L for red cell folate (13). ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. aviam iatracellulare.
celiac sprue biopsy specimens showed typical flattened villus morphology. Villus heights in biopsy specimens from all HIV-infected patients were within 2SD of our normal mean of 400 pm, although values from 5 biopsy specimens were < 300 km (Figure 3). Crypt depth was greater than the normal mean of 150 Frn in 7 patients and was greater than the mean + 2SD in 3 patients. The villus height to crypt depth ratio was less than the normal mean of 2.7:l.O in all patients and less than the mean -2SD in 7 patients. The percentage of mitotic figures was greater than the normal mean of 2.0% in 8 patients and greater than the upper range of control in 3 patients (Figure 3).
Compared with findings in biopsy specimens from normal controls, cytospectrophotometric analysis of the villus tips showed significantly enlarged enterocyte nuclei in 8 and a decreased number of nuclei in 6 of the 9 HIV-infected patients (Figure 4). The mean enterocyte nuclear size in biopsy samples from HIV patients was approximately 1.5 times larger than the enterocyte nuclear size in the control biopsy samples (P < 0.001). The density of enterocyte nuclei was lower in HIV-infected biopsy specimens than in control biopsy specimens (P < 0.001). Mean values for both measurements in celiac biopsy specimens were significantly different from controls (P < 0.05) but similar to the HIV-infected patients. No significant differences in cytospectrophotometric measurements were seen between patients with ARC and AIDS.
Human immunodeficiency virus RNA was detected by in situ hybridization in 5 of 9 patient jejunal biopsy specimens. All 5 HIV-infected biopsy samples showed evidence of chronic inflammation. Human immunodeficiency virus infection was identified in 4-6 enterocytes per section in villi of one patient who had normal circulating T-helper cells but abnormal cytospectrophotometry, steatorrhea, and lactose intolerance (Figure 5). Most enterocytes did not support
0 4
4 9 OO’
.,.,. :.,j:.,. .:..:::::::::::::: ....,., ...,: ..................................v.... ....,., ..............
3
600-
:
300-
3.0 -
s L
. .r
c E
i.0
200 400- i
F
0
Villus
g E
100
.:
U.’ : ?? . .
2
$ijjijjijjj$ijlj B
%
0
. :.:. 0 : :. ::.:lj I;;_;t: . .. . :, : I, .:,.
VilluslCrypt
Mitotic
Index
i
Figure 3. Villus height, crypt depth, villus length to crypt depth ratios, and mitotic indices of jejunal biopsy specimens from HIV-infected patients. Patient values for villus and crypt measurements are means of 3-10 well-oriented villi per biopsy sample. The normal ranges (shaded bars) represent means ?2SD of 93 measurements from 10 normal specimens. The mitotic index is the number of mitotic figures in a minimum of 300 crypt epithelial cells per biopsy specimen and is expressed as a percentage for each biopsy specimen. The normal range for mitotic index is based on measurements from 10 normal biopsy specimens. ?? , ARC; El, ARC + G. lamblia; 0, AIDS: 0, AIDS + M. aviam iatracellulare.
5 F 9
:: ................ 3 0
9 5 52. c
0 2
+
Nuclear
Size
;.i;ii;;;:;:<
........l../....//.. ....i...\......\ .........../\.. .\............/.... ..\.I...\../.... :... ....I.....\\ :.:. .:.:. :*:x;; ::. .: .../...I.. .
8 z
5
5
0 ‘i 0
depth
“E i
1
1.0
200 ~II~U
Crypt
.
. 2.0
w
0 height
4.0 9-
Vol. 100, No. 6
II
. 00
1
Nuclear
4
0-l density
Figure 4. Cytospectrophotometric measurements of enterocyte nuclear size and density (number of nuclei per 100 km) in jejunal biopsy specimens from HIV-infected and celiac sprue patients and control subjects. Individual values are the mean of 100 measurements from each biopsy sample, using enterocytes located in the top third of the villus. Significant differences were found between HIV-infected and control biopsy samples for both enterocyte nuclear size (34 k 0.3 vs. 22 2 0.2 urn’; P < 0.001) and enterocyte nuclear density (20 k 1.7 vs. 30 k 3.2 nuclei/100 pm; P < 0.001). Mean values in celiac sprue biopsy specimens for both nuclear size (37 2 5.6) and density (18 f 2.5) were significantly different from controls (P < 0.05) but not different from HIV-infected patients. Shaded bars indicate normal ranges. ?? , ARC; 0, ARC + G. lamblia; 0, AIDS; 0, AIDS + M. avium intracellulare.
June 1991
HIV INFECTION
OF JEJUNUM
1525
Figure 5. The presence of HIV RNA detected by in situ hyhridization in villus enterocytes of a patient with ARC. The patient had been diagnosed as seropositive for HIV 1 month before our evaluation and had a Z-month history of diarrhea. The CD4-CD8 ratio was normal (1.5) with 426 T-helper cells/mn?. Severe fat malabsorption (23% of dietary intake) and lactose intolerance (55 ppm H, over baseline) with negligible lactase activity were found. Villus length to crypt depth ratio (2.l:l.O) and mitotic index (1.52%) were both within our normal ranges, whereas the mean enterocyte nuclear size was above normal at 33 pm’. A.
Mid infected
villus of jejunal biopsy specimen enterocytes (original magnification
B. Higher magnification intensity of HIV RNA X1.250: bar = 10 wm).
showing two HIVx500; bar = 20 p,m).
of Figure 5A demonstrating high-signal in enterocytes (original magnification
C. Different field from same biopsy specimen showing another infected enterocyte with adjacent intraepithelial mononuclear cell (original magnification X1250; bar = 10 km).
viral replication to the same degree as the mononuclear cells, as evidenced by the need for a longer exposure time for detection (2 weeks vs. 5 days) and by reduced density of silver grains within the cells. Schmorl’s stain for argentaffin granules (22) identified enterochromaffin cells in serial sections, all separate from HIV-infected cells. The remaining four positive biopsy specimens contained HIV-infected mononuclear cells in the intraepithelial spaces, the subepithelium, the lamina propria, and near the crypt base (Figure 6). Jejunal biopsy specimens from uninfected control subjects were negative for HIV RNA. Positive and negative signals were obtained from HIV-infected and uninfected cultured T cells (CEM line). No positive cells were detected following RNAse treatment of tissue, with hybridization with pSP64 vector alone or with a probe to the simian immunodeficiency virus, indicating that positive signals were specific for HIV RNA.
Discussion The present study confirms that intestinal malabsorption can occur in an early stage and may be an important cause of malnutrition in AIDS. Jejunal mucosal HIV infection in our patients was associated with altered enterocyte differentiation and function, as shown by reversal of the villus length to crypt depth ratio, increased enterocyte nuclear size, and depression of digestive enzyme activity. The absence of other identifiable intestinal pathogens suggests a direct role for HIV in the pathogenesis of AIDS enteropathy and the potential for antiviral therapy directed at the mucosal lesion. Others have demonstrated HIV infection in the intestine by in situ hybridization (7-9) and immunohistochemical (5) techniques. Infected enterochromaffin cells, lymphocytes, and macrophages have been identified in rectal biopsy specimens (7,9,23),
1526
HEISE ET AL.
Figure 6. HIV RNA in two subepitbelial mononuclear cells in the jejunal mucosa of a patient with AIDS (original magnification X 1250; bar = 10 pm).
and two studies (7,8) suggest that rectal crypt epithelial cells are capable of infection with HIV. In jejunal biopsy specimens, HIV antigen was found previously in lamina propria mononuclear cells and in occasional intraepithelial lymphocytes (5). Our data provide the first evidence of jejunal enterocyte infection by HIV. The finding of direct enterocyte infection with HIV in one biopsy specimen and lamina propria mononuclear cell infection in four others suggests a wider presence of undetected HIV in the jejunal mucosa. The method of in situ hybridization for RNA identifies cells that are actively expressing viral nucleic acid but does not detect cells in a latent stage of infection. Although it is not known whether the CD4 receptor is present on enterocytes, the CD4 receptor RNA was expressed in the HT29 colon carcinoma cell line and in normal colon cells (24). Two colon carcinoma cell lines (HT29 and SW480) supported productive HIV infection in vitro (24), and HIV was shown to replicate in normal and malignant intestinal cell cultures (25). In another tissue, renal tubule and glomerular epithelial cells were found capable of infection by HIV in vivo (26). The route of jejunal mucosal infection by circulating blood or direct contact with HIV in the intestinal lumen cannot be determined by our studies. Previous studies reported nonspecific inflammation, partial villus atrophy (1,5), and increased number of intraepithelial lymphocytes (1,27) in the intestinal mucosa of jejunal biopsy samples from patients with AIDS. A study of mucosal abnormalities in HIV-infected patients documented increased crypt depth and decreased mitoses together with decreased
GASTROENTEROLOGY
Vol. 100, No. 6
lactase activity (5). Another study of jejunal biopsy specimens in ambulatory HIV-infected patients described increased jejunal villus to crypt ratio with increased mitoses (28). In our study of early infection, the villus length to crypt depth ratio was reversed equally in AIDS and ARC biopsy samples, and increased numbers of mitoses were found in three of nine patients. Among the studies, the difference in observed mitotic indices may relate to duration and severity of the disease, so that mitoses are more likely to be decreased in hospitalized patients at later stages of AIDS. Altered cell proliferation could result in functionally immature enterocytes with enlarged nuclei, as has been described in the mucosal disease tropical sprue (29). The finding by cytospectrophotometry that enterocyte nuclear size was increased and the numbers of enterocytes per unit villus length were decreased equally in both ARC and AIDS (Figure 4) suggests that immature enterocytes with larger nuclei are produced at early stages of infection. Enterocyte immaturity could account for the decreased expression of brush border enzymes in all our patients and for generalized malabsorption of both dietary fat and orally administered n-xylose observed in about half the patients. The finding of steatorrhea in all patients with ARC and the most severe fat malabsorption in a patient with HIV-infected enterocytes underscores the relationships among mucosal HIV infection, enterocyte immaturity, and dysfunction. Finding a uniform incidence of low lactase levels in the HIV-infected patients is not surprising because this enzyme is less abundant than sucrase in the intestinal mucosa and hence more susceptible to significant inhibition. Nevertheless, finding a similar ratio of sucrase to lactase in HIV-infected patients and controls suggests nonselective suppression of brush border enzyme activity. Finding low jejunal lactase in all patients, but lactose intolerance by the breath hydrogen test in only 4 patients, suggests that the enzymatic defect precedes its clinical manifestation. Finding levels of brush border folate hydrolase below our published norm (19) in half of the patients (Figure 2) is consistent with the generalized effect of HIV infection on brush border hydrolases. The correlation of low brush border folate hydrolase with red cell folate levels (Figure 2) suggests a regulatory effect of this enzyme on the supply of folate to tissues. Although evidence for HIV infection of the jejunal mucosa is now quite convincing, the pathogenesis of the observed mucosal change is speculative. Possible explanations include retarded cell maturation due to low mucosal folate, a direct effect of HIV on enterocyte function, or an indirect cytokine-mediated effect on cell proliferation and maturation.
June
1991
HIV INFECTION
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Standard methods of clinical chemistry. Volume II. New York: Academic, 1958:34-39. Roe JH, Rice EW. A photometric method for determination of free pentoses in animal tissues. JBiol Chem 1948;173:507-512. Newcomber AD, McGill DB. Thomas PJ, Hoffman AF. Prospective comparison of indirect methods for detecting lactase deficiency. N Engl J Med 1975;293:1232-1235. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biothem 1968;22:99-107. Halsted CH, Beer WH, Chandler CJ, Ross K. Wolfe BM, Bailey L, Cerda JJ. Clinical studies of intestinal folate conjugases. J Lab Clin Med 1986;107:228-232. Goldstein E, Donovan RM, Kim Y. Applications of computerized microscopic image analysis in infectious diseases. Rev Infect Dis 1988;10:92-102. Brahic M, Haase AT. Detection of viral sequences of low reiteration frequency by jn situ hybridization. Proc Nat1 Acad Sci 1978;75:6125-6129. Perls M. Nachweis von Eisenoxyd in Oewissen Pigmenten. Virchows Arch Path01 Anat 1867;39:42-48. Levy JA, Margaretten W, Nelson J. Detection of HIV in enterochromaffin cells in the rectal mucosa of an AIDS patient. Am J 1989;84:787-789. Gastroenterol Adachi A, Koenig S, Gendelman HE, Daugherty D, Gatton-Celli S, Fauci AS, Martin MA. Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus. J Virol 1987;61:209-213. Moyer MP, Ramirez A, McKee-Suarez MS, Gendelman HI. In: Sixth International Conference on AIDS. HIV infection of gastrointestinal (GI) cell cultures (abstr). Volume II. San Francisco: 1990:140. Cohen AH, Sun NCJ, Shapshak P, Imagawa DT. Demonstration of human immunodeficiency virus in renal epithelium in HIV-associated nephropathy. Mod Path01 1989:2:125-128. Weber JR, Dobbins WO. The intestinal and rectal epithelial lymphocyte in AIDS. Am J Surg Pathol 1986;10:627-639. Cummins AG, LaBrooy JT, Stanley DP, Rowland R, Shearman DJC. Quantitative histological study of enteropathy associated with HIV infection. Gut 1990;31:317-321. Swanson VL, Thomassen RW. Pathology of the jejunal mucosa in tropical sprue. Am J Path01 1965;46:511-551.
Received April 11, 1990. Accepted November 2, 1990. Address requests for reprints to: Charles H. Halsted, M.D., Division of Clinical Nutrition and Metabolism, TB 156, University of California, Davis, California 95616. The authors acknowledge the technical expertise of David Vinson, Carol Chandler, Rich Peterson, Dale J. Martfeld, and Robert Munn. Supported by grants from the University of California AIDS Task Force; from the National Institutes of Health, DK-35747 and R29-NS27338; and from the Bristol-Myers Squibb Company.