JOURNAL OF PATHOLOGY, VOL.
161: 1 19-127 (1990)
ULTRASTRUCTURE OF THE JEJUNAL MUCOSA IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION MINNIE M. MATHAN*, G. E. GRIFFIN?, A. MILLER?, P. BATMAN?, S. FORSTER:,
A. PINCHING: AND W. HARRIS:
*The Wellcome Research Unit, Christian Medical College Hospital, Vellore. India; TDepartments of Communicable Diseases and Histopathology, St George’s Hospital Medical School, London, U.K.; $Departments of Medicine, Clinical Immunology and Genito-urinary Medicine, St Mary’s Hospital Medical School, London, U.K. Received 6 January 1989 Accepted 22 January 1990
SUMMARY The ultrastructural changes in the jejunal muscosa of 11 male patients, three with clinical AIDS, five with AIDS related complex-progressive generalized lymphadenopathy (ARC-PGL), and three who were only HIV antibody positive, were studied. In the enterocytes,major abnormalities were proliferation of smooth endoplasmic reticulum mitochondria1changes, vacuolization of cells, and fat hold up. In the lamina propria, degeneration of enteric nerve axons and smooth muscle were seen. Microvasculatureshowed both endothelialcell degeneration and hyperplasia.The presence of tubuloreticularinclusions inendothelialcells paralleled the stage of the disease. Since none of the 11patients had any opportunisticinfection, these changes are likely to be the effect of HIV infection. KEY WORDS-HIVinfection,acquired immune deficiency syndrome,jejunal biopsy, ultrastructure.
INTRODUCTION
infected with HIV, none of whom had evidence of other viral, bacterial, or parasitic infection of the gut. We present ultrastructural evidence to show that HIV infection is associated with striking abnormalities, both in the enterocytes and in several of the lamina propria constituents.
In patients with the acquired immunodeficiency syndrome (AIDS), chronic diarrhoea and malabsorption can lead to marked weight loss as indicated by the local name ‘slim disease’ in Africa.’ Although nucleotide probing has demonstrated incorporation of the human immunodeficiency virus (HIV) genome into crypt enterocytes,2 it is still not clear whether MATERIALS AND METHODS HIV infection by itself causes a primary lesion in the intestinal mucosa. The histopathologic changes Jejunal biopsies were obtained with Crosby capreported in the small and large bowel epithelium sules under fluoroscopic control, from the proximal in patients with AIDS3-’ have been attributed to jejunum from 1 1 HIV antibody-positive male homoopportunistic infections, inflammation, and neo- sexual subjects, with diarrhoea of more than 2 plasia, and do not fully explain the pathogenesis of weeks’ duration, attending HIV outpatient clinics in the diarrhoea.6 In some patients with AIDS, chronic London. Three of these patients had AIDS based on diarrhoea and malabsorption are present with- CDC criteria,* four patients had AIDS related comout any identifiable enteric infectious agent.’ We plex (ARC), one patient had persistent generalized therefore studied the ultrastructural morphology of lymphadenopathy (PGL), and three of the subjects jejunal biopsies from 11 adult homosexual males were asymptomatic except for mild intermittent diarrhoea. Opportunistic gastro-intestinal infections Addressee for correspondence:Minnie M. Mathan, Wellcome were excluded at the time of biopsy by repeated Research Unit, CMC Hospital, Vellore 632004, India. examination of wet smears of stools, appropriate 0022-3417/90/060119-09 $05.00 0 1990 by John Wiley & Sons, Ltd.
120
M. M. M A T H A N ET AL.
Fig. I+a) Apical part of the epithelial cell from upper third of villus with marked proliferation of smooth endoplasmic reticulum (SER) (asterisks).Periphery of these hyperplastic areas show dense aggregates within the lumen of SER (arrow). x 28 000.(b) Higher magnification to show structures resembling tubuloreticular inclusion (TRI) within dilated endoplasmic reticulum (arrows). x 67 000
stool cultures, and examining paraffin-embedded tissue with the modified Ziehl Neelsen stain for acid fast organisms, PAS for fungi, and Giemsa and Gram stains for protozoa and bacteria. The biopsies were rapidly retrieved and a portion was immediately fixed in 2.5 per cent glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy, and another portion was fixed in 10 per cent formol saline for light microscopic examination.’ The biopsies were post-fixed in osmium tetroxide, enblock stained in uranyl acetate. and embedded in epoxy resin. Sections were cut on a LKB ultratome IV with a diamond knife and stained with lead citrate. The biopsies were coded and the electron microscopist was unaware of the HIV antibody status or clinical classification of the patients. Control jejunal biopsies were obtained from ten male subjects, not belonging to accepted high-risk categories for acquiring HIV infection, in whom enteric infection had been excluded by methods identical to those used in the HIV-positive patients. The light microscopic appearance of these jejunal biopsies was normal. The HIV status of the controls was not tested because of ethical consideration.
The study was approved by the Ethical Committees of the St George’s and St Mary’s Hospitals, London and written consent was obtained from the patients. RESULTS The biopsies from the HIV-infected patients showed a variety of changes in the epithelium and lamina propria, in striking contrast to the biopsies from controls, which were within normal limits when examined by the electron microscope. Epithelium Proliferation of the smooth endoplasmic reticulum (SER) was a striking feature in all but one (asymptomatic) subject (Fig. la). In enterocytes on the upper third of the villi, the SER occupied up to half the area of the cell. Electron-dense inclusions with occasional tubular structures were present as clusters of varying sizes within dilated tubules of endoplasmic reticulum at the periphery of the area of SER proliferation (Fig. Ib). These inclusions
121
JEJUNAL MUCOSA IN HIV INFECTION
Table I-Epithelial changes
Clinical grading Asymptomatic Asymptomatic Asymptomatic ARC ARC ARC ARC
PGL AIDS AIDS AIDS
Fat absorption*
Fat in enterocytes
26 52 28 34 27
257
-
44
117
32 39 40 52
158 190 58 209 68
Age
44
180
124 127 64
Fat in lamina propria
SER proliferation
TRI Mitochondria1 Cell in SER change vacuolization
++
-
-
++ ++ +++
+++
+++ +++ +++
+ ++ ++ + ++ + ++ ++
++ -
++ ++ ++ -
+ +
+ +++ ++ ++
-
+
~~
*Trioleinbreath test normal > 180. SER = Smooth endoplasmicreticulum;TRI =tubuloreticular inclusion.
were present in all the patients with AIDS and in one patient with ARC, but were not found in any of the three asymptomatic individuals. SER proliferation and the presence of inclusions did not correlate with the presence of fat particles in enterocytes (Table I). There was a reduction in the amount of rough endoplasmic reticulum with dropping off of ribosomes. The mitochondria were decreased in number, particularly in the enterocytes on the upper third of the villi. They were altered in shape with branching, irregular, or elongated forms as well as C- or cupshaped mitochondria (Fig. 2). The mitochondria1 cristae were longitudinally placed and the matrix appeared dense. In patients with ARC, PGL, and AIDS, these changes were present in 30-60 per cent of the mitochondria of cells in the upper third of the villi (Table I). Large vacuoles which were occasionally empty or contained membranous or proteinaceous debris were present in enterocytes in the upper and middlethird of the villi in half the subjects. A thin lining membrane could be visualized in some vacuoles (Fig. 3). Throughout the villus and crypt epithelium there were scattered, single, or small groups of enterocytes which appeared to be degenerated, with wellpreserved adjacent cells. The degenerated cells were darker staining with clumping of nuclear chromatin dilated rough endoplasmic reticulum and damaged mitochondria. There was an increase in lysosomal structures in most of the enterocytes on the villi.
Paneth cells of four patients showed alteration in the morphology of granules, with some of them showing crystalline inclusions (Fig. 4). Intra-epithelial lymphocytes with signs of activation were also increased. Fat droplets were present in the apical cytoplasm, within the smooth endoplasmic reticulum, Golgi region, in dilated intercellular spaces, and beneath the basement membrane in the majority of patients (Table I). In four subjects, the intercellular spaces were widely dilated and contained lipid material with a linear configuration. Lamina propria
Plasma cells, macrophages, eosinophils, and mucosal mast cells contributed to the increased cellularity of the lamina propria in all the patients. The endoplasmic reticulum of plasma cells was dilated and many appeared to be undergoing cytolysis. Lysosomal particles were prominent in the macrophages, but viral particles could not be identified. Degranulation of eosinophils and mucosal mast cells was also observed. Scattered pyknotic and apparently degenerated cells were seen throughout the lamina propria, but the precise identification of the cell type was difficult. Blood vessels were prominent and appeared to be increased in number. The endothelial cell cytoplasm contained tubuloreticular inclusions (TRIs), which were more prominent in patients with AIDS. The endothelial cells of some of the blood vessels showed
I22
M. M. MATHAN E T A L .
Fig. 3-Enterocyte with vacuoles (V) in the cytoplasm distorting the nucleus. Membranous lining present in the vacuoles in the lower part. x 6000
Fi_e 2-Enterocyte from the upper third of villus containing mitochondria with altered shape. longitudinal cristae and dense matrix x 21 500
a varying degree of degenerative changes with marked swelling of the cells occluding the lumen and loss of cellular organelles, with dilatation of RER and swelling and lossofcristaeofmitochondria (Fig. 5). Scattered endothelial cells were necrotic. Other vessels showed a hyperplastic response with an increase in cellular organelles, Weibel-Palade bodies. and multivesicular bodies. The nuclei of these cells were enlarged. more irregular, and contained prominent nucleoli. The basement membrane appeared thickened or reduplicated. Often small vessels with occluded lumens were found near crypts with focal degeneration of enterocytes. In all the patients. changes in the autonomic nerve fibrils of the intrinsic nerve plexus of the lamina propria of the intestinal mucosa were quite striking and have been reported in detail elsewhere." Briefly. there was proliferation of the nerve fibres and extensive degeneration of axons, which were
swollen and electron-lucent with loss of cellular organelles. Schwann cells showed an increase in Iipofuschin pigment (Table 11). Myofibrillar degeneration of smooth muscle cells in the lamina propria was a striking feature. The periphery of the cells showed large protrusions of sarcoplasmic membrane in between dense plaques. Degenerated axonal bundles were often found adjacent to such damaged smooth muscle fibres (Fig. 6 ) . DISCUSSION The abnormalities detected in the epithelium and Iamina propria of the jejunal mucosa of these patients, without evidence of opportunistic infection. suggest that HIV infection itself can damage this tissue. HIV genome incorporation has been demonstrated by nucleotide probing in jejunal and rectal mucosa.' The failure to detect HIV particles" in any of these biopsies may be because of a low rate of production of morphologically recognizable
123
JEJUNAL MUCOSA IN HIV INFECTION
Fig. G P a n e t h cell showing altered granules and dilated endoplasmic reticulum.
particles or difficulty in distinguishing the particles from damaged cellular organelles. In v i m HIV can infect epithelial cells derived from colorectal carcinoma in continuous culture.” In villous enterocytes. proliferation of the SER was a striking feature. Proliferation of the SER has been reported in enterocytes and liver cells secondary to drug-induced enzyme production. 13.’4 These patients were not on any drugs. in particular Azidothymidine or barbiturates. during the month prior to the biopsy. nor were they consuming alcohol in excess. SER proliferation can also occur during active fat absorption.” but these patients had been fasting for at least 7 h prior to biopsy. The presence of fat particles in the epithelium and lamina propria indicates derangement of fat transport but the prominent SER proliferation. not correlated with intracellular lipid. is not found in other malabsorptive conditions such as tropical sprue.“ The
x 17 OOO
nature of the particles seen within the SER is not clear and some of these could be lipoprotein particles. However. the prevalence of particles with a tubular configuration within the SER mainly in patients with AIDS suggests that these could be associated specifically with HIV infections. A variety of tubular complexes have been described in viral infections and neoplasia. ‘*.I9 Morphologic abnormalities in mitochondria with abnormal orientation of cristae have been reported in association with alteration of mitochondrial enzymes following exposure to toxins. neoplasia. hypoxia. and delayed fixation.” Delayed fixation is an unlikely cause of the mitochondrial alterations, since the control biopsies with normal mitochondria were processed in exactly the same fashion. The mitochondrial abnormalities in these patients are likely to be from the alterations responsible for SER proliferation. although hypoxia
’’
124
M. M. MATHAN ET A L .
Fig. 5-Part of the cross-section of a capillary with swollen endothelial cells occluding the lumen (L). TRIs (T) are present in endothelial cell cytoplasm. Basement membrane (arrow) shows reduplication.
secondary to microvascular alterations may also play a part. Scattered damage and necrosis of enterocytes was maximal in patients with AIDS, confirming reports by others.” This enterocyte damage is due to cellmediated cytolysis of virus-infected cells,” with an additional contribution by ischaemia due to vascular occlusion. The Paneth cell alteration could be secondary to malabsorption of nutrients, especially zinc, as has been observed in other gastro-intestinal disorders.” Striking abnormalities were seen in the lamina propria. The involvement of the nerves to a similar extent in all the groups of patients (Table 11) suggests that this tissue may be one of the primary targets of the virus.” The enteric nervous system is important in controlling blood flow, motility, and epithelial transport. The neural damage in the intrinsic plexuses can artly explain the diarrhoea in patients with AIDS.2 B
x
29 600
The smooth muscle myofibrillar degeneration and prominent sarcoplasmic protrusions are similar to the changes described in isolated smooth muscle fibres in vitro, as a result of electrical or agonist stim~lation.’~ Such changes have not been reported previously in intact smooth mucles. These sarcoplasmic protrusions are similar to the sarcolemmal pads occurring during myofibrillar degeneration of striated muscle secondary to denervation, deprivation of vascular supply, metabolic insufficiency, cachexia, mechanical compression, drug action, or muscle disorders.” In the present group of patients, the smooth muscle abnormalities are most likely secondary to denervation caused by damage to the intrinsic autonomic plexus. Motility abnormalities consequent on this smooth muscle damage may also contribute to diarrhoea. The endothelial abnormalities and the reduplication of the vascular basement membrane26in the lamina propria confirm that the endothelium is
J k J U K A L Y U C O S A I \ HI\'IUFECTIOS
Table 11-Changes Clinical grading Asymptomatic Asymptomatic Asymptomatic ARC ARC ARC ARC
PG L AIDS AIDS AIDS
in the lamina propria
Swelling
Endothelial changes Hyperplasia
++ + + + + ++ ++ + +t '++ ++
++ ++ + + + +++ +++ + + ++
TRI
+ -+ + -r+
-
++ +++ ++
Axonal degeneratbon
Smooth muscle degeneration
++ ++ +++ ++ ++ +++ ++ ++ +++ ++
++ ++ ++ ++ + ++ + + +++
-+++
t
TRI = Tubuloreticular inclusion
Fig 6-A cross-section(C) and longitudindl seclion ( L )of smooth muscle cells with sarcoplasmicmembrane projections (P)and adjacent non-myehndtednerve (N)with axondl degeneration x 15 000
126
M. M. MATHAN ET AL.
another tissue which is affected early by HIV. endothelial degeneration and necrosis were present, no virus particles were found in endothelial cells or in ad,acent macrophages, as described in neCrOtiZing vasculitis of peripheral nerves in AIDS.27Prevalence of TRI paralleled the disease stage (Table 11) as observed by other workers also.28 The proliferation and hyperplasia of the endothelium are similar to pre-Kaposi changes Seen in skin capi11anes.29'30 This is of si@cance, since gut mucosa is the most common site of visceral Kaposi's sarcoma in AIDS.3' Two possible factors explain the pathogenesis of fat malabsorption in these patients. Metabolic alterations in the enterocytes, as evidenced by the SER and mitochondria1 changes, may interfere with triglyceride synthesis in enterocytes. The linear arrays in lateral intercellular spaces of enterocytes and lamina propria may be the product of this alteration. Additionally, the failure of pumping action due to smooth muscle and enteric nerve axonal damage may be an important factor, as evidenced by the empty and 'ymphatics9 with large amounts of fat held up in the lamina propna
paces.^"^^ ACKNOWLEDGEMENTS
The Wellcome Research Unit is supported by the Trust, London in association with the Christian Medical College, Vellore. G.E.G. is a Wellcome Trust Senior Lecturer. This work was supported in part by the Medical Research Council, U.K. REFERENCES 1. SeWadda D. MugeWa RD. Sewankambo NK. el 01. Slim di-:
2.
3. 4.
5.
6. 7.
a new disease in Uganda and its association with HTLV-III infection. Lancer 1985; 2: 849-852. Nelson JA, Wiley CA. Reynolds-Kohler C, RCCSC CE. Margaretten W. Levy JA. Human immunodeficiency virus detected in bowel epithelium from patients withgastrointestinalsymptoms.Lancet 1988; I: 259-262. Rotterdam H, Sommers SC. Alimentary tract biopsy lesions in the acquired immune deficiency syndrome. Parhology 1985; 17: 181-192. Sewankambo N, MW""a RD, Goodgame R, e f al. EnteroPathic AIDS in Uganda. An endoscopic, histological and microbiological study. AIDS 1987; 1:9-13. Dobbins W O 111. Weinstein WM. Electron microscopy ofthe intestine and rectum in acquired ImmUnodCficienCysyndrome. Gastroenrerdog) 1985: 88: 738-749. Dworkin B, Wonnser GP, Rosenthal WS, er al. Gastrointestinal manifestations of the acquired immunodeficiency syndrome: a review of 22 cases. Am J Gasooenrerol1985;80: 774-778. Kotler DP. Gaetz HP. Lange M, Lange M, Klein EB. Holt PR. Enteropathy associated with the acquired immunodeficiency syndrome. Ann Infern Med 1984; 101: 421428.
8. Centen for Disease Control. U.S.Department of Health and Human Services, Atlanta, Georgia. Classification system for human Tlymphotropic virus type III/lymphadenopathy-associatedvirus infection. Ann Infern Med 1986; 105 236237. 9. Batman PA, Miller ARO, Forster SM, Harris JRW, Pinching AJ, Griffin GE. Jejunal enteropathy associated with human immunodeficiency virus infection: quantitative histology. J Ciin Parhol1989; 42275-281. 10. Griffin GE, Miller A, Batman P, er al. Damage to jejunal intrinsic autonomic nerves in human immunodeficiency virus infection. AIDS 1988;2: 379-382. I I . Munn RJ, Marx PA, Yamamoto JK, Gardner MB. Ultrastructural comparison of the retroviruses associated with human and simian acquired immunodeficiency syndromes. Lob Invesr 1985; 53: 194-199. 12. Adachi A, Koenig S, Gendelman HE, er al. Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus. J Virol1987;61: 209-213. 13. Thomas FB, Baba N, Greenberger NJ, Salsburey D. Erect of phenobarbitalonsmallintatinal structureand function in therat.JLab Ciin Med 1972;80: 548-558. 14. Barka T, Popper H. Liver enlargement and drug toxicity. Medicine 1967;46:103-117. 15. Sabcsin SM, F r a a A. Electron microscopic studies of the assembly. intracellular transport, and secretion ofchylomicrons by rat intestine. /Lipid Res 1977; 18: 49651 I . 16. Mathan M,Mathan VI, Baker SJ. An electron microscopic study of jejunal mucosal morphology in control subjects and in patients with tropical sprue in southern India. Gastroenrerology 1975: 68:17-32. 17. Tytgat GN, Rubin CE, Saunders DR. Synthesis and transport of lipoprotein particles by intestinal absorptive cells in man. JClin Invesr 197 I ;50: 2065-2078. 18. Pfeifer U,Thomscn R, Legler K, er al. Experimental non-A, non-B hepatitis: four typcs of cytoplasmic alteration in hepatocytes of inVirchows Arch BICell Parhol] 1980; 33: 233-243. fected chimpan-. 19. U m a n BG, Saito H,Kasac M.Tubular arrays in the endoplasmic reticulum in human tumorcells. Lub Invest 1971; t(:492498. 20. Ghadially FN. Ultrastructural Pathology of the Cell and Matrix. 2nd ed.London: Buttmvorths, 1982: 164-167. 21. Kotler DP, Weaver SC, Terzakis JA. Ultrastructural features of epithelial cell degeneration in rectal crypts of patients with AIDS. Am J Surg Parhol1986; 10: 531-538. 22. W e k r JR, Dobbins WO."he intcstinal and mtalepithelial lymphocytein AIDS. Am JSurg h h o i 1986; 10:627-639. 23. Kobayashi Y, Suzuki H, Konno f,Tada K, Yamamoto TY. Ultrastructural alterations of Paneth cells in infants associated with gastrointestinal symptoms. Tohokv J € x p Med 1983; 13%225-2311. 24. Lundgrcn 0,Svanvik J, Jivegard L. Enteric nervous system. I . Physiology and pathophysiology of the intestinal tract. Dig D k Sci 1989;34: 264-283. 25. Maruyama I, Kobayashi M, Yoshida C, Momose K. Ultrastructure ofsinglesmooth musclecellscontracted by carbachol and calcium ion. J Pharmacobio Dyn 1987; 10: 396403. 26, V m k o R,~~l lamina rafold anatomy and significance for maintenance oforderly tissue stNctuTcs,Am Jparhol 1974;TI: 316346. 27. Gherardi R, Lebargy F, Gaulard P, Mhiri C, Bemaudin JF, Gray F. ~ ~ ~ ~ varulitis t i and z HIV i ~replication ~ in peripheral nerves, N ~~~iJ Med 1989; 321: 68-86. 28. Kostianovsky M, k n s t e i n JM, k h a f z, G~~~~~PM, Cytomembranous inclusions observed in acquired immunodeficiency synArch parho[ Lob Med 1987; dromc, clinical and 111: 218-223. 29. McNutt NS. Fletcher V, Conant MA.Early lesions ofKaposi's a r coma in homosexual men.An ultrastructural companson with other vascular proliferations in skin. Am JPathol1983; 111: 62-77. 30. Kunu AA, Gelderblom HR, Winkel T,Reichart PA, Ultrastructural findings in oral Kaposi's Sarcoma (AIDS). J oralpatho[ 1987; 1 6 372-379. 31. Friedman SL, Wright TL, Altman DF. Gastrointestinal Kaposi's sarcomainpatientswith acquired immunodeficiency syndrome. Endoscopicand autopsy findings. Getrroenrerology 1985; 89: 102-108. 32. McHale NG, Roddie IC. The effect of transmural pressure on p u m p ing activity in isolated bovine lymphatic vessels. J Physiol 1976; 261: 255-265.
JUUNAL MUCOSA IN HIV INFECTION 33. Alessandrini C. Gerli R. Sacchi G, Ibba L, Pucci AM, Fruschelli C. Cholinergic and adrenergic innervation of mesenterial lymph vessels in guinea pig. Lymphology 1981: 1 4 1-6.
127
34. McHale NG, Roddie IC. Thornbury KD. Nervous modulation of spontaneous contractions in bovine mesenteric lymphatics. J Physiol
1980; 309:461-471.
161: 1 19-127 (1990)
ULTRASTRUCTURE OF THE JEJUNAL MUCOSA IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION MINNIE M. MATHAN*, G. E. GRIFFIN?, A. MILLER?, P. BATMAN?, S. FORSTER:,
A. PINCHING: AND W. HARRIS:
*The Wellcome Research Unit, Christian Medical College Hospital, Vellore. India; TDepartments of Communicable Diseases and Histopathology, St George’s Hospital Medical School, London, U.K.; $Departments of Medicine, Clinical Immunology and Genito-urinary Medicine, St Mary’s Hospital Medical School, London, U.K. Received 6 January 1989 Accepted 22 January 1990
SUMMARY The ultrastructural changes in the jejunal muscosa of 11 male patients, three with clinical AIDS, five with AIDS related complex-progressive generalized lymphadenopathy (ARC-PGL), and three who were only HIV antibody positive, were studied. In the enterocytes,major abnormalities were proliferation of smooth endoplasmic reticulum mitochondria1changes, vacuolization of cells, and fat hold up. In the lamina propria, degeneration of enteric nerve axons and smooth muscle were seen. Microvasculatureshowed both endothelialcell degeneration and hyperplasia.The presence of tubuloreticularinclusions inendothelialcells paralleled the stage of the disease. Since none of the 11patients had any opportunisticinfection, these changes are likely to be the effect of HIV infection. KEY WORDS-HIVinfection,acquired immune deficiency syndrome,jejunal biopsy, ultrastructure.
INTRODUCTION
infected with HIV, none of whom had evidence of other viral, bacterial, or parasitic infection of the gut. We present ultrastructural evidence to show that HIV infection is associated with striking abnormalities, both in the enterocytes and in several of the lamina propria constituents.
In patients with the acquired immunodeficiency syndrome (AIDS), chronic diarrhoea and malabsorption can lead to marked weight loss as indicated by the local name ‘slim disease’ in Africa.’ Although nucleotide probing has demonstrated incorporation of the human immunodeficiency virus (HIV) genome into crypt enterocytes,2 it is still not clear whether MATERIALS AND METHODS HIV infection by itself causes a primary lesion in the intestinal mucosa. The histopathologic changes Jejunal biopsies were obtained with Crosby capreported in the small and large bowel epithelium sules under fluoroscopic control, from the proximal in patients with AIDS3-’ have been attributed to jejunum from 1 1 HIV antibody-positive male homoopportunistic infections, inflammation, and neo- sexual subjects, with diarrhoea of more than 2 plasia, and do not fully explain the pathogenesis of weeks’ duration, attending HIV outpatient clinics in the diarrhoea.6 In some patients with AIDS, chronic London. Three of these patients had AIDS based on diarrhoea and malabsorption are present with- CDC criteria,* four patients had AIDS related comout any identifiable enteric infectious agent.’ We plex (ARC), one patient had persistent generalized therefore studied the ultrastructural morphology of lymphadenopathy (PGL), and three of the subjects jejunal biopsies from 11 adult homosexual males were asymptomatic except for mild intermittent diarrhoea. Opportunistic gastro-intestinal infections Addressee for correspondence:Minnie M. Mathan, Wellcome were excluded at the time of biopsy by repeated Research Unit, CMC Hospital, Vellore 632004, India. examination of wet smears of stools, appropriate 0022-3417/90/060119-09 $05.00 0 1990 by John Wiley & Sons, Ltd.
120
M. M. M A T H A N ET AL.
Fig. I+a) Apical part of the epithelial cell from upper third of villus with marked proliferation of smooth endoplasmic reticulum (SER) (asterisks).Periphery of these hyperplastic areas show dense aggregates within the lumen of SER (arrow). x 28 000.(b) Higher magnification to show structures resembling tubuloreticular inclusion (TRI) within dilated endoplasmic reticulum (arrows). x 67 000
stool cultures, and examining paraffin-embedded tissue with the modified Ziehl Neelsen stain for acid fast organisms, PAS for fungi, and Giemsa and Gram stains for protozoa and bacteria. The biopsies were rapidly retrieved and a portion was immediately fixed in 2.5 per cent glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy, and another portion was fixed in 10 per cent formol saline for light microscopic examination.’ The biopsies were post-fixed in osmium tetroxide, enblock stained in uranyl acetate. and embedded in epoxy resin. Sections were cut on a LKB ultratome IV with a diamond knife and stained with lead citrate. The biopsies were coded and the electron microscopist was unaware of the HIV antibody status or clinical classification of the patients. Control jejunal biopsies were obtained from ten male subjects, not belonging to accepted high-risk categories for acquiring HIV infection, in whom enteric infection had been excluded by methods identical to those used in the HIV-positive patients. The light microscopic appearance of these jejunal biopsies was normal. The HIV status of the controls was not tested because of ethical consideration.
The study was approved by the Ethical Committees of the St George’s and St Mary’s Hospitals, London and written consent was obtained from the patients. RESULTS The biopsies from the HIV-infected patients showed a variety of changes in the epithelium and lamina propria, in striking contrast to the biopsies from controls, which were within normal limits when examined by the electron microscope. Epithelium Proliferation of the smooth endoplasmic reticulum (SER) was a striking feature in all but one (asymptomatic) subject (Fig. la). In enterocytes on the upper third of the villi, the SER occupied up to half the area of the cell. Electron-dense inclusions with occasional tubular structures were present as clusters of varying sizes within dilated tubules of endoplasmic reticulum at the periphery of the area of SER proliferation (Fig. Ib). These inclusions
121
JEJUNAL MUCOSA IN HIV INFECTION
Table I-Epithelial changes
Clinical grading Asymptomatic Asymptomatic Asymptomatic ARC ARC ARC ARC
PGL AIDS AIDS AIDS
Fat absorption*
Fat in enterocytes
26 52 28 34 27
257
-
44
117
32 39 40 52
158 190 58 209 68
Age
44
180
124 127 64
Fat in lamina propria
SER proliferation
TRI Mitochondria1 Cell in SER change vacuolization
++
-
-
++ ++ +++
+++
+++ +++ +++
+ ++ ++ + ++ + ++ ++
++ -
++ ++ ++ -
+ +
+ +++ ++ ++
-
+
~~
*Trioleinbreath test normal > 180. SER = Smooth endoplasmicreticulum;TRI =tubuloreticular inclusion.
were present in all the patients with AIDS and in one patient with ARC, but were not found in any of the three asymptomatic individuals. SER proliferation and the presence of inclusions did not correlate with the presence of fat particles in enterocytes (Table I). There was a reduction in the amount of rough endoplasmic reticulum with dropping off of ribosomes. The mitochondria were decreased in number, particularly in the enterocytes on the upper third of the villi. They were altered in shape with branching, irregular, or elongated forms as well as C- or cupshaped mitochondria (Fig. 2). The mitochondria1 cristae were longitudinally placed and the matrix appeared dense. In patients with ARC, PGL, and AIDS, these changes were present in 30-60 per cent of the mitochondria of cells in the upper third of the villi (Table I). Large vacuoles which were occasionally empty or contained membranous or proteinaceous debris were present in enterocytes in the upper and middlethird of the villi in half the subjects. A thin lining membrane could be visualized in some vacuoles (Fig. 3). Throughout the villus and crypt epithelium there were scattered, single, or small groups of enterocytes which appeared to be degenerated, with wellpreserved adjacent cells. The degenerated cells were darker staining with clumping of nuclear chromatin dilated rough endoplasmic reticulum and damaged mitochondria. There was an increase in lysosomal structures in most of the enterocytes on the villi.
Paneth cells of four patients showed alteration in the morphology of granules, with some of them showing crystalline inclusions (Fig. 4). Intra-epithelial lymphocytes with signs of activation were also increased. Fat droplets were present in the apical cytoplasm, within the smooth endoplasmic reticulum, Golgi region, in dilated intercellular spaces, and beneath the basement membrane in the majority of patients (Table I). In four subjects, the intercellular spaces were widely dilated and contained lipid material with a linear configuration. Lamina propria
Plasma cells, macrophages, eosinophils, and mucosal mast cells contributed to the increased cellularity of the lamina propria in all the patients. The endoplasmic reticulum of plasma cells was dilated and many appeared to be undergoing cytolysis. Lysosomal particles were prominent in the macrophages, but viral particles could not be identified. Degranulation of eosinophils and mucosal mast cells was also observed. Scattered pyknotic and apparently degenerated cells were seen throughout the lamina propria, but the precise identification of the cell type was difficult. Blood vessels were prominent and appeared to be increased in number. The endothelial cell cytoplasm contained tubuloreticular inclusions (TRIs), which were more prominent in patients with AIDS. The endothelial cells of some of the blood vessels showed
I22
M. M. MATHAN E T A L .
Fig. 3-Enterocyte with vacuoles (V) in the cytoplasm distorting the nucleus. Membranous lining present in the vacuoles in the lower part. x 6000
Fi_e 2-Enterocyte from the upper third of villus containing mitochondria with altered shape. longitudinal cristae and dense matrix x 21 500
a varying degree of degenerative changes with marked swelling of the cells occluding the lumen and loss of cellular organelles, with dilatation of RER and swelling and lossofcristaeofmitochondria (Fig. 5). Scattered endothelial cells were necrotic. Other vessels showed a hyperplastic response with an increase in cellular organelles, Weibel-Palade bodies. and multivesicular bodies. The nuclei of these cells were enlarged. more irregular, and contained prominent nucleoli. The basement membrane appeared thickened or reduplicated. Often small vessels with occluded lumens were found near crypts with focal degeneration of enterocytes. In all the patients. changes in the autonomic nerve fibrils of the intrinsic nerve plexus of the lamina propria of the intestinal mucosa were quite striking and have been reported in detail elsewhere." Briefly. there was proliferation of the nerve fibres and extensive degeneration of axons, which were
swollen and electron-lucent with loss of cellular organelles. Schwann cells showed an increase in Iipofuschin pigment (Table 11). Myofibrillar degeneration of smooth muscle cells in the lamina propria was a striking feature. The periphery of the cells showed large protrusions of sarcoplasmic membrane in between dense plaques. Degenerated axonal bundles were often found adjacent to such damaged smooth muscle fibres (Fig. 6 ) . DISCUSSION The abnormalities detected in the epithelium and Iamina propria of the jejunal mucosa of these patients, without evidence of opportunistic infection. suggest that HIV infection itself can damage this tissue. HIV genome incorporation has been demonstrated by nucleotide probing in jejunal and rectal mucosa.' The failure to detect HIV particles" in any of these biopsies may be because of a low rate of production of morphologically recognizable
123
JEJUNAL MUCOSA IN HIV INFECTION
Fig. G P a n e t h cell showing altered granules and dilated endoplasmic reticulum.
particles or difficulty in distinguishing the particles from damaged cellular organelles. In v i m HIV can infect epithelial cells derived from colorectal carcinoma in continuous culture.” In villous enterocytes. proliferation of the SER was a striking feature. Proliferation of the SER has been reported in enterocytes and liver cells secondary to drug-induced enzyme production. 13.’4 These patients were not on any drugs. in particular Azidothymidine or barbiturates. during the month prior to the biopsy. nor were they consuming alcohol in excess. SER proliferation can also occur during active fat absorption.” but these patients had been fasting for at least 7 h prior to biopsy. The presence of fat particles in the epithelium and lamina propria indicates derangement of fat transport but the prominent SER proliferation. not correlated with intracellular lipid. is not found in other malabsorptive conditions such as tropical sprue.“ The
x 17 OOO
nature of the particles seen within the SER is not clear and some of these could be lipoprotein particles. However. the prevalence of particles with a tubular configuration within the SER mainly in patients with AIDS suggests that these could be associated specifically with HIV infections. A variety of tubular complexes have been described in viral infections and neoplasia. ‘*.I9 Morphologic abnormalities in mitochondria with abnormal orientation of cristae have been reported in association with alteration of mitochondrial enzymes following exposure to toxins. neoplasia. hypoxia. and delayed fixation.” Delayed fixation is an unlikely cause of the mitochondrial alterations, since the control biopsies with normal mitochondria were processed in exactly the same fashion. The mitochondrial abnormalities in these patients are likely to be from the alterations responsible for SER proliferation. although hypoxia
’’
124
M. M. MATHAN ET A L .
Fig. 5-Part of the cross-section of a capillary with swollen endothelial cells occluding the lumen (L). TRIs (T) are present in endothelial cell cytoplasm. Basement membrane (arrow) shows reduplication.
secondary to microvascular alterations may also play a part. Scattered damage and necrosis of enterocytes was maximal in patients with AIDS, confirming reports by others.” This enterocyte damage is due to cellmediated cytolysis of virus-infected cells,” with an additional contribution by ischaemia due to vascular occlusion. The Paneth cell alteration could be secondary to malabsorption of nutrients, especially zinc, as has been observed in other gastro-intestinal disorders.” Striking abnormalities were seen in the lamina propria. The involvement of the nerves to a similar extent in all the groups of patients (Table 11) suggests that this tissue may be one of the primary targets of the virus.” The enteric nervous system is important in controlling blood flow, motility, and epithelial transport. The neural damage in the intrinsic plexuses can artly explain the diarrhoea in patients with AIDS.2 B
x
29 600
The smooth muscle myofibrillar degeneration and prominent sarcoplasmic protrusions are similar to the changes described in isolated smooth muscle fibres in vitro, as a result of electrical or agonist stim~lation.’~ Such changes have not been reported previously in intact smooth mucles. These sarcoplasmic protrusions are similar to the sarcolemmal pads occurring during myofibrillar degeneration of striated muscle secondary to denervation, deprivation of vascular supply, metabolic insufficiency, cachexia, mechanical compression, drug action, or muscle disorders.” In the present group of patients, the smooth muscle abnormalities are most likely secondary to denervation caused by damage to the intrinsic autonomic plexus. Motility abnormalities consequent on this smooth muscle damage may also contribute to diarrhoea. The endothelial abnormalities and the reduplication of the vascular basement membrane26in the lamina propria confirm that the endothelium is
J k J U K A L Y U C O S A I \ HI\'IUFECTIOS
Table 11-Changes Clinical grading Asymptomatic Asymptomatic Asymptomatic ARC ARC ARC ARC
PG L AIDS AIDS AIDS
in the lamina propria
Swelling
Endothelial changes Hyperplasia
++ + + + + ++ ++ + +t '++ ++
++ ++ + + + +++ +++ + + ++
TRI
+ -+ + -r+
-
++ +++ ++
Axonal degeneratbon
Smooth muscle degeneration
++ ++ +++ ++ ++ +++ ++ ++ +++ ++
++ ++ ++ ++ + ++ + + +++
-+++
t
TRI = Tubuloreticular inclusion
Fig 6-A cross-section(C) and longitudindl seclion ( L )of smooth muscle cells with sarcoplasmicmembrane projections (P)and adjacent non-myehndtednerve (N)with axondl degeneration x 15 000
126
M. M. MATHAN ET AL.
another tissue which is affected early by HIV. endothelial degeneration and necrosis were present, no virus particles were found in endothelial cells or in ad,acent macrophages, as described in neCrOtiZing vasculitis of peripheral nerves in AIDS.27Prevalence of TRI paralleled the disease stage (Table 11) as observed by other workers also.28 The proliferation and hyperplasia of the endothelium are similar to pre-Kaposi changes Seen in skin capi11anes.29'30 This is of si@cance, since gut mucosa is the most common site of visceral Kaposi's sarcoma in AIDS.3' Two possible factors explain the pathogenesis of fat malabsorption in these patients. Metabolic alterations in the enterocytes, as evidenced by the SER and mitochondria1 changes, may interfere with triglyceride synthesis in enterocytes. The linear arrays in lateral intercellular spaces of enterocytes and lamina propria may be the product of this alteration. Additionally, the failure of pumping action due to smooth muscle and enteric nerve axonal damage may be an important factor, as evidenced by the empty and 'ymphatics9 with large amounts of fat held up in the lamina propna
paces.^"^^ ACKNOWLEDGEMENTS
The Wellcome Research Unit is supported by the Trust, London in association with the Christian Medical College, Vellore. G.E.G. is a Wellcome Trust Senior Lecturer. This work was supported in part by the Medical Research Council, U.K. REFERENCES 1. SeWadda D. MugeWa RD. Sewankambo NK. el 01. Slim di-:
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