Lymph Node Modification in Patients with the Acquired Immunodeficiency Syndrome (AIDS) or with AIDS Related Complex (ARC)

Path. Res. Pract. 180, 590-611 (1985)

Lymph Node Modification in Patients with the Acquired Immunodeficiency Syndrome (AIDS) or with AIDS Related Complex (ARC) A Histological, Immuno-Histopathological and Ultrastructural Study of 45 Cases J. Diebold*; CI. Marche**; J. Audouin*; J. P. Aubert*; A. Le Tourneau*, CI. Bouton**; M. Reynes*; J. Wizniak**; F. Capron*; V. Tricottet*

* Service Central "J. Delarue" d'Anatomie et de Cytologie pathologiques (Prof. J. Diebold), H6tel-Dieu de Paris ** Laboratoire d'Anatomie et de Cytologie Pathologiques (Dr. CI. Marche), H6pital Claude Bernard

SUMMARY The authors present the results of a histopathological study on the lymph-nodes taken from 45 subjects suffering from either an AIDS or from a chronic adenopathy corresponding to the definition of AIDS related complex (ARC). The various aspects observed were classed as type I to type IV. The lymph-node modifications observed in the 29 patients with an ARC could be divided into three principle groups: an extensive follicular hyperplasia associated with other elementary lesions or type IA (25 lymph nodes from 23 patients); changes resembling a multicentric Castleman syndrome or type IB (1 case); angioimmunoblastic-like (AIL) lesions or type II (2 cases) and an association of lesions of type II (7 lymph-nodes from 6 patients). During AIDS, the adenopathy usually disappears, and the small lymph-nodes removed, especially on autopsy, show an extensive lymphoid depletion (type III) with systematic sclerosis (15 lymph-nodes from 14 patients). When adenopathy persists, it is due to infections complications (tuberculosis, cryptococcosis, avian mycobacteriosis and Whipple's disease like lesions). Of the 10 patients in whom a Kaposi's sarcoma was observed, only 6 showed lymphnode involvement, or type IV. The different histopathological lesions seem to appear according to an evolving succession, proven by certain association of lesions and by successive biopsies. In our series, 17% of subjects with an ARC evolved to AIDS. Lymph-node biopsy allows a possible ARC to be implicated on the association of the following simple lesions: follicular hyperplasia with partial or total destruction of the perifollicular lymphocytic cisterna, infiltration of the germinative centres by streams of small lymphocytes, evolving to an aspect of a "burst" germinative centre and various sinusal reactions with, in particular, the presence of neutrophilic polynuclear cells. The biopsy also allows the forms with bad prognosis to be recognized: those with AIL-like aspect or multicentric Castleman-like syndrome, which seems to represent a particular 0344-0338/8510180-0590$3.5010

© 1985 by Gustav Fischer Verlag, Stuttgart

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 591

evolutive form. Finally, it also detects, in certain cases, the localization of a Kaposi syndrome, signalling the passage to AIDS. The immunopathological studies present a double interest. Firstly, they offer arguments in favour of the diagnosis: increase in the number of T8 lymphocytes in the germinative centres with the formation of small clusters and disruption of the network of dendritic reticular cells, and the inversion of the T41T8 ratio in the extra-follicular cortical regions, by either a decrease in T4 lymphocytes or by an increase in T8 lymphocytes. Secondly, it allows a better understanding of the physiopathology of the disease: polyclonal B-lymphocyte hyperplasia with increase in the number ofT8lymphocytes, and histiocytic hyperplasia, then decrease of the T4lymphocytes and, finally, progressive lymphoid depletion with sclerosis. The ultrastructural study detects several intracytoplasmic inclusions. The 2 types the most interesting are the test-tube and ring-shaped forms (TRF) and the tubulo-reticular structures (TRS). The first, have a certain diagnostic interest for human pathology and should be regarded carefully. The second, which can be observed in other human pathological states, seem to be linked to a production or a local reaction of interferon. However above all, this study allowed viral particles to be detected, which resemble retroviruses, in the spaces surrounded by dendritic reticular cells in the hyperplastic germinative centres in subjects having an ARC. It thus seems to us indispensable to carry out a lymph-node biopsy in all subjects at risk for AIDS who present a polyadenopathy for more than 3 months. An immunohistopathological study on frozen tissue and an ultrastructural study should always be carried out to complete the routine histopathological study in patients with chronic polyadenopathy and at risk for AIDS.

Introduction The acquired immunodeficiency syndrome, or AIDS, is a severe dysimmunitary state occuring in certain categories of individuals2: homosexuals 60, 62, 63, habitants or emigrants from the Cariban (Haiti) and Equatorial Africa (Zaire)l4, intravenous drug users, and transfused subjects or hemophiliacs havin received coagulants coming from high risk populations lf. The "Groupe de Travail Fran<;ais sur Ie SIDA" (The French Work-Group on AIDS) defined the patient with AIDS as: "a patient below 60 years of age, having no acute infection and undergoing no treatment susceptible to causing an immune depression, and having one or several opportunistic infections and/or a Kaposi sarcoma,,42. Apart from these patients having confirmed AIDS, there exists a particular state manifested essentially by persistant adenopathyl, 9, 20, 22, 26, 28, 29, 43, 49, 50, 58-60, 62, 63, 87. This lymphadenopathical syndrome, or LAS, was initially defined by the "Center for Disease Control" 11. More recently, a more strict definition has been adopted by the Work Group of the National Institute of Health USA (NIH) to separate the different states characterised by polyadenopathy and susceptible to occur in subjects at high-risk. The clinical symptomatology of this "AIDS related complex" or ARC can include the following differWe thank Mr. Wolfelsperger for the photographs. This study was supported in part by grants from Broussais-H6tel Dieu Faculty of Medicine Scientific Council and in part by grants from the "Fondation pour la Recherche Medicale".

ent signs which must exist for at least 3 months: lymphadenopathy in at least 2 extra-inguinal sites; loss of at least 7 kg or 10% body weight; intermittent or continuous fever at 38° or higher; diarrhea, asthenia, and night sweats. The biological symptoms can consist of the following: a reduction in the number of helper T4 cells in the peripheral blood; a decrease in the ratio of T4 helper to T8 cytotoxic/suppressor lymphocytes; an anemia, or leucopenia, thrombopenia, lymphopenia; a polyclonal serum hypergammapathy; reduction of the blastogenic response of the lymphocytes to mitogens; a cutaneous anergy; and circulating immune complexes. A patient is considered as having an ARC when he presents at least 2 clinical symptoms and 2 biological signs. The significance of LAS and ARC is interpreted differently. For some, they are states completely different from AIDS, and for others, real prodromal forms of AIDS evolving to AIDS in time varying from several weeks to several months, or even years. However, these states are susceptible to regression2,26. We report here the histopathological, immunohistopathological and ultrastructural aspects of lymphatic lymph-nodes taken by biopsy or autopsy on 16 patients with AIDS and 29 high-risk patients, all answering the definition of ARC. Material and Methods This study involved 37 males, including 30 homosexuals, and 8 heterosexual females. Among the risk factors, apart from

592

J. Diebold et al.

Table 1. Patients with lymphadenopathy syndrome (LAS) or AIDS-related complex (ARC) Lymph node Year of Case Sex Age at Risk Histopathol. biopsy factors biopsy Locali- Size zation type 1 M EUC.

30

2 M RIL.

23

3 M FOR.

28

4 M BAC. 5 M PRI.

20

6 F DAG

M 7 LAC.

46 27

24

cerv.

0.7 em

foIl. hyperpl.

-

cerv.

1 em

foIl. hyperpl.

-

N

homo. Mar. 1982 May drug addict 1983 homo. Apr. Haitian 1983

cerv.

1 em

foIl. hyperpl.

-

"'-

Nov. 1980

cerv.

± 1 em

foIl. hyperpl. and AIL-like

-

+

N

1982

cerv.

± 1 em

foIl. hyperpl.

+

+

N

Feb. 1983

Haitian Oct. 1980 Feb. 1981 homo. Aug. 1983 Oct. 1983

a axill. b cerv.

1 em

foIl. hyperpl. ND

+

"'-

1.2 em

AIL-like

1 em

foIl. hyperpl. ND foIl. hyperpl. ND and AIL-like

a ing. b ing.

M

22

homo. July drug 1983 addict

cerv.

M 9 AP.. 10 M CHA

25

homo. July 1983 homo. Apr. 1983 Sept. 1983

axill.

8

11 F PIG 12 M MOT. 13 M JOU.

44

40 26 26

14 M CHA. 15 M LAP.

35

16 M LOU.

34

36

+

T4/T8 Begin. of the disease

homo. Apr. 1983 homo. Apr. 1983

1 em 1 em

X..

HTLV LAV

N

Sept. 1982 Dec. 1981

"'ND

N

+

"'-

Apr. 1983

"'-

Sept. 1982

1 em

foIl. hyperpl. ND

"'-

0.7 em 1 em

foIl. hyperpl. AIL-like Kaposi

Jan. 1983 1982

cerv.

1 em

foIl. hyperpl. ND

cerv;

1 em

foIl. hyperpl. ND

axill.

1 em

foIl. hyperpl. ND and AIL-like

+

"'-

homo. Oct. 1983 homo. Jan. 1983

axill.

1.2 em

foIl. hyperpl. ND

+

ND

cerv.

1 em

foIl. hyperpl.

homo. Apr. 1983

cerv.

1.5 em

Castleman like

Aug. drug addict 1983 homo. July 1983 homo. July 1983

5 months after the biopsy (Sept. 1983): HTLV +, LAV +

1969

foIl. hyperpl. ND

a cerv. b cerv.

Evolution

+

+

"'-

+

"'-

1981

ND

Jan. 1983 1982

pneumocystis carinii pneumonia

5 months after the 2nd biopsy: generalized Kaposi's sarcoma May 1984: Kaposi's sarcoma - CMV (alveolar washing) Multiple infectious disease

3 months later: Kaposi's sarcoma, infectious disease (AIDS) Death: 1984

Sept. 1983 Lymphadenopathies and thrombopenia since 1982

ND

ND

ND

Thrombocytopenia Kaposi's sarcoma of the skin a few weeks after lymph node biopsy multiple infectious disease Death: July 1983

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 593 Table 1. Patients with lymphadenopathy syndrome (LAS) or AIDS-related complex (ARC) Lymph node Year of Case Sex Age at Risk Histopathol. biopsy factors biopsy Locali- Size zation type 17 M DOU.

24

homo. June 1983 Dec. 1983

18 M DHI. M 19 BEN.

36

M 20 ASS.

23

21 M RIT.

19

26

homo. June 1983 Nov. drug addict 1983

a cerv. axil!. 1.5 em b cerv. 0.8 to 2cm ing. 1.5 em cerv.

cerv. occip.

March a drug addict 1983 cerv. b Sept. 1983 ing. homo. Oct. 1983

HTIV LAV

foil. hyperpl. ND

N

March 1983 Aug. 1983

no further information

March 1983

wife and baby have AIDS

1 em

foil. hyperpl. NO

ND

ND

1 em

foil. hyperpl. NO

NO

'\

1.5 cmfoll. hyperpl. NO

NO

'\

NO

N

Aug. 1983

NO

'\

Oec.1983

a axill.

2cm

foil. hyperpl.

b cerv.

0.6 em

foil. hyperpl.

-

33

homo. Nov. 1983

axill.

2cm

foil. hyperpl.

23 M LER. 24 M OAe.

26

drug June addict 1984 homo. Oct. 1983

cerv.

1.7 em

foil. hyperpl. NO and AIL-like

cerv.

0.8 em

foil. hyperpl.

neutropenia and thrombopenia since 1982

40 M DOD.

25

homo. July drug 1983 addict

mg.

1.2 em

foil. hyperpl. NO and AIL-like

no res. NO avail. rhumatoid factor

Apr. 1983

41

M

15

homo. Nov. drug 1983 addict

axill.

1.5 em

foil. hyperpl. NO

dubi- N ous posit.

May 1983

43

F

27

Zairian Jan. 1984

cerv.

1 em

foil. hyperpl. ND and AIL-like

+

N

July 1983

44 F JAG. 45 F PEe.

22

drug Feb. addict 1984 drug Apr. addict 1984

axill.

2.5 em

foil. hyperpl. NO

NO

ND

axill.

2cm

foil. hyperpl. NO

NO

N

TEL FUL.

27

Evolution

foil. hyperpl. and AIL-like

M 22 YAY.

37

T4/T8 Begin. of the disease

homosexuality, 11 of the patients in this series were intravenous drug-users (including 4 homosexuals), 11 patients of Haitian origin (5 homosexuals) or who had visited that country. Finally, this series included 3 subjects originating from Zaire (Tables 1 and 2). Sixteeri patients (12 men, 4 women) were seen at the stage of confirmed AIDS. Lymph-node were studied 14 times at autopsy and 3 times on biopsy, one of the patients (no. 25) having had a biopsy of a cervical lymph-node 13 months before his death, answering at that time the definition of ARe. Thirty-five lymph-nodes were taken from the 29 patients having an ARC (24 men, 5 women), 6 patients having two successive biopsies.

May 1984: fever, asthenia, polyadenopathie splenomegaly lymph node biopsy: same aspect

Histopathological study All lymph-nodes were studied after fixation in neutral formalin or in aqueous Bouin's solution, inclusion in paraplast and staining of the sections with hematein-eo sin-safran, PAS stain, Giemsa, silver impregnation according to Gordon-Sweet. Ziehl stain, Gram stain, and Grocott stain were performed in all cases where the presence of an etiological agent was suspected.

Immunohistopathology Immunotyping study was carried out on frozen sections in 12 cases (no. 7 b, 9, 16, 18,21,22,24,34 and 40 to 43). It was also

J. Diebold et al.

594

Table 2. Patients with AIDS Case Sex Age at Risk Year of Lymph node biopsy factors biopsy Locali- Size Histopathol. zation type 25

VED.

M

39

homo. Oct. 1981

a cerv.

autop. b Nov. med. 1982

1 em

AlL-like (LAS)

2 em

depletion Kaposi

2 em

HTLV LAV

T4/T8 Begin.

of the disease

Evolution

July 1981

Death: Nov. 1982

'\

Nov. 1982 LAS +

Death: Apr. 1983

+

'\

+

+

'\

depletion acute tuberculosis

ND

ND

26 M GAU.

38

homo. autop. mesent. 1983

27 M ALE.

39

< 1cm Haitian autop. med. hetero. 1983 mesent.

depletion Kaposi

+

+

'\

Oct. 1982 LAS +

Death: Feb. 1983

28 F ALE.

31

Haitian autop. med hetero. 1982

< 1 em

depletion

ND

ND

ND

Jan. 1981 no LAS

Neurotoxoplasmosis Death: Feb. 1982

29

24

Haitian autop. a < 1 em hetero 1983 med. b mesent < 1 em

depletion Kaposi

+

'\

July 1981 no LAS

Death: Feb. 1983

ND

ND

Feb. 1983

Death: July 1983

N

Nov. 1982

Necrotizing colitis Death: July 1983

MAR.

30 DIS.

F

depletion Whipple-like

M

56

homo. autop. mesent. 1983

31 M FAU.

29

32 M COM.

38

depletion ND homo. autop. mesent. < 1 em 1983 drug addict 1.5 em depletion homo. autop. mesent. + histiocytosis Haitian 1983 with M. avium

33 M SAM.

35

homo. autop. mesent. < 1 em African 1983

depletion

ND

+

'\

Apr. 1983 no LAS

Death: July 1983

34 M KOZ.

32

Zairian biopsy axill. hetero Sept. 1983

1 em

foil. hyperpl. ND and AIL-Like

+

'\

June 1983 LAS +

Multiple infectious disease Still alive, but in bad condition

35 M GUA.

36

homo. autop. lat. 1983 aort.

< 1 em

depletion Kaposi

ND

36 M CAY.

29

homo. May mesent. < 1 em 1983 laparo.

depletion

+

37 M FOU.

33

homo. autop. mesent. < 1 em 1983

depletion

ND

38

M

42

Haitian autop. mesent. hetero 1983

depletion crytococcus

ND

39 ELI.

F

24

Zairian autop. mesent. < 0.5 em depletion hetero 1983

42

M

24

homo. biopsy cerv. Dec. 1983

KAR.

GIR.

1 em

2 em

Kaposi

1.5 em AIL-like

LAS

'\

+

LAS

+

+

Death: 1983

Death: Sept. 1983

'\

Dec. 1980 LAS +

Kaposi's sarcoma of the skin Death: Aug. 1983

'\

June 1982 LAS +

Kaposi's sarcoma of the skin Death: 1983

+

'\

1981 Death: Sept. 1983 LAS + in July 1983

+

+

'\

ND

+

'\

LAS1979 LAS +

Baby with AIDS Death: Oct. 1983 Multiple infectious disease in 1983 (AIDS) Still alive in May 1984

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 595 done on fixed paraffin embedded sections on 20 lymph nodes taken from 17 patients with ARC (no. 1 to 5, 6 a and b, 7 a and b, 8, 10 a and b, 15 to 19, 21, 22 and 24) and on 3 lymph-nodes taken from 2 patients with AIDS (no. 25, 34). For technical reasons, no valid result was obtained with lymph-node 7 a and no account was taken of this specimen. Eleven monoclonal antibodies were used on 4 mu frozen sections: T4 (Coulter clone), OKT6, OKT8, OKT11, OKM1 (Orthoclone), J3D3 (Dr. Kazatchkine and Boucheix, Paris) for C3 b receptors, HLA-Dr and 2B markers: Alb 6 and Alb g (Dr. Boucheix, Paris), IgD (Dakopatts). KiM4 (Dr. Feller, Radzun and Prof. Lennert, Kiel) against reticular dendritic cells. The antigens were revealed either by an indirect immunofluorescent study (Goat anti-mouse FITC EY) or by a three stage immunoperoxidase staining technique (Rabbit anti-mouse peroxidase, Swine anti-rabbit peroxidase, Dakopatts). The immunoglobulin heavy and light chains were detected by a technique of direct immunofluorescence either on frozen sections or on paraffin sections using a technique previously described17 with A, G, M, kappa and lambda FITC antiserum (Pasteur). Histiocytic enzymes markers (lysozyme, alpha-I-antitrypsin, alpha-l-antichymotrypsine, Dakopatts) were revealed on paraffin sections of 18 lymph nodes (no. 1 to 4, 6 to 8, 10, 16, 18,20 to 22, 24, 25, 29,34,36) with the Sternberger PAP method.

1.1. Lymphadenopathy with predominant lymphoid follicular hyperplasia (type IA) This aspect was observed in 25 lymph-nodes from 23 patients. The follicular hyperplasia was made up of voluminous, round, oval or pyriform follicles. Sometimes they were confluent, forming arcs. The germinative centres contained mostly large cells with basophilic cytoplasm (centroblasts, immunoblasts) with frequent mitosis. Several plasmocytes could be associated, with sometimes Russel bodies. Histiocytes were numerous, isolated or grouped in small nests, or looking like tingible bodies macrophages. The arrangement of the germinative centres into dark and light hemispheres was often easily recognizable. The perifollicular lymphocytic mantle zone was sometimes normal and well-defined. However, more often a variable number of follicles had lost their lymphocytic mantle zone (Fig. 1). This mantle zone sometimes disappeared completely or sometimes only on a part of the circumference. The centroblasts and centrocytes were then only separated from the cells of the perifollicular lymphoid parenchyma by dendritic reticular cells.

Ultrastructural methods Samples of 11 lymph-nodes were fixed in 1.5% glutaraldehyde in PBS buffer, pH 7.4, and post-fixed with 2% osmium tetroxide for standard ultrastructural study. Nine were from patients with ARC (no. 2, 7, 9, 16, 18,21 b, 22, 24, 41), and two were from patients with AIDS (no. 34 and 36). Two lymph-nodes fixed in Bouin's solution and embedded in paraffin from 2 patients with AIDS (cases 29 and 32) exhibiting lymphnode diffuse histiocytosis were studied at the ultrastructural level after paraffin removal in xylene, rehydration, postfixation with osmium tetroxide and embedding in epoxy resin.

Results The different histopathological aspects observed with the lymph-nodes taken from the two groups of patients will be successively studied, and then the immunohistopathological and ultrastructural results for each group will be given.

1. Histopathological aspects of lymph-nodes during

ARC

This group of 29 patients consisted of 24 men and 5 women. Among the men, 17 were only homosexual (including one Haitian), 3 homosexual and drug-users, 4 drug addicts. Of the women, 2 were from Haiti (including one drug addict), a third was from Zaire, and the 2 others were drug addicts (Table 1). In these patients, three types of lesions could be described as being at the origin of the lymphadenopathy: lymphadenopathy with predominant follicular hyperplasia (type IA); multicentric Castleman-like lymphadenopathy (type IB); and angio-immunoblastic-like lymphadenopathy (AIL-like) (type II).

Fig. 1. Case no. 3. ARC type IA. Follicular hyperplasia with large germinal center. No lymphocytic mantle zone. The sharp delineation of the germinal center is destroyed by a cluster of small lymphocytes with irregular nuclei. Vascular hyperplasia in the interfollicular zone. Giemsa. G = 225 x. Inset: WarthinFinkeldey giant cell. Giemsa. G = 600 x.

596 .

J. Diebold et al.

In all cases, an infiltration of the germinative centres by small lymphocytes was associated with this disappearance of the perifollicular mantle zone. This infiltration occurred at the periphery of the germinative centres with disappearance and destruction of their external limit, presenting a "nibbled" or "mott-eaten" aspect (Fig. 1). The infiltration also occurred along the arterial vessels and the capillaries, either in the form of only a few lymphocytes in single file, or in the form of numerous cells in dark bands clearly cutting the cellular population of the germinative centres. This infiltration masked the structure of the germinal centres or had the aspect of a displaced or burst follicle (Fig. 2). The morphology of these lymphocytes must be emphasized. Their cytoplasm was pale, the nuclei irregular with very dense clumps of heterochromatin. Finally, in 6 cases, multinucleate cells of Warthin Finkeldey type were discovered in several germinative centres (Fig. 1). Modifications of the deep cortical area and interfollicular zones were associated with this follicular hyperplasia. The surface area of these T dependant areas was normal or increased (22 lymph-nodes). The cellular population was constituted by a mixture, in variable quantities, of small

Fig. 2. Case no. 21. ARC type IA. Hyperplastic follicle disrupted by a heavy lymphocytic infiltration of the germinal centre. HES. G = 150 x.

lymphocytes with round nuclei or irregUlar nuclei more or less convoluted. A variable number of macrophages containing cell debris, eosinophilic granulocytes and mast cells were dispersed among the lymphocytes. Interdigitating reticular cells could be detected in 19 lymph-nodes due to their large pale cytoplasm and their clear drawn-out nucleus with a very irregular outline. In most cases, these areas were also infiltrated by centroblasts and immunoblasts forming the aspect of a large polymorphous hyperplasia. Sometimes, it seemed that these cells with basophilic cytoplasm originated from the germinative centres. Moreover, in the deep cortical and especially interfollicular areas, an extensive vascular hyperplasia was always observed, predominantly involving the epithelioid venules (Fig. 1). Endothelial cells, more numerous than usual, stood out in the light. A very active transparietal lymphocytic traffic was visible; the vascular walls were not thickened by PAS positive deposits, however, sometimes a collagen sclerosis ensheathed these vessels. In several lymph-nodes, oedematous zones with localised lymphoid depletion (cases 3, 13, 15) were discovered, with slight haemorrhagic exsudates. Finally, in other cases, small loci of systematic sclerosis were noted (cases 11, 13, 15, 34). A hyperplasia of the plasmocytoid T lymphocytes was stated in 10 cases. This took the form of nests of lymphoid cells of average size with a light nucleus, containing small nucleoli. On Giemsa staining, these nests appeared as pale blue zones, often situated in contact with the epithelioid venules in the interfollicular zones at the limit of the deep cortical area or in contact with the interfollicular or subcapsullary sinuses. These cells had a pale cytoplasm, greyblue, ovoid, excentric with regard to the nucleus, hence their plasmocytoid aspect. A plasmocytic hyperplasia was alway observed. The medullar cords contained almost uniquely mature plasmocytes with a few proplasmocytes as well as immunoblasts which were sometimes plasmoblastic. Numerous plasmocytes were also discovered in the most exterior cortex under the marginal sinus. Nests of plasmocytes were often situated around the interfollicular vessels or in deep cortical areas. Let us recall the presence of plasmocytes in the germinative centres. In most of the cases, the extent of this plasmocytosis was directly proportional to the extent of the immunoblastic hyperplasia of the lymph-node parenchyma. A parenchymatous histiocytic hyperplasia was seen in all lymph-nodes. The extent and expression were variable. Sometimes it could consist of small masses of macrophagic histiocytes along the marginal and interfollicular sinuses (18 lymph-nodes). Nests or small granuloma of histiocytic cells, of epithelioid cells and of cells intermediate between these two types were recognized between the follicles, often at their direct contact in 21 lymph-nodes, forming an aspect quite similar to that found in toxoplasmosis or syphilis. The sinusal reactions were very diverse, different aspects being associated in the marginal and interfollicular sinuses of the same lymph-node. In all the cases studied, lympho-

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 597

cytes with round or irregular nuclei and cytophagic histiocytes, particularly erythrophagic or containing PAS positive hyalin corpuscles (Hamasuchi-Wesenburg bodies) were seen in most of the cortical sinuses. Associated with these, in nearly every case, were granulocytes, mostly neutrophilic. In 4 cases having the above characteristics, lymphocytes with a more or less foliate nucleus with several large cells ressembling interdigitating reticular cells were also associated. An immature sinusal histiocytosis, corresponding in fact to a type B lymphocytosis with distension of certain sinusal segments, was noted in interfollicular or sub-capsular sinuses in 20 lymph-nodes. An accumulation of macrophagic histiocytes considerably distended certain sinusal segments in 2 lymph-nodes. Finally, an intense active congestion of the small arterial vessels in the conjunctival lamellae with venous distension was observed in half of the lymph-nodes studied. A peri-adenitis consisting of an infiltration due to a mixture of lymphocytes and plasmocytes was also noted in most of the cases studied.

A

1.2. The lymphadenopathies resembling an angiofollicular polyadenopathy with plasmocytosis or multicentric Castleman syndrome (type IB) Only one patient presented this aspect. The lymph-node was the site of a follicular hyperplasia, but the germinative centres were less voluminous. Centrocytes and centroblasts were few, whilst the dendritic reticular cells were hyperplasic and organised in concentric sheets forming the aspect of an onion bulb (Fig. 3). The vessels were larger than normal, with thick walls (Fig. 3). Hyalin deposits could be found in these centres. They were associated with atrophy of the deep cortical areas by lymphoid depletion with agglomeration of the interdigitating reticular cells (Fig. 3). The vascular hyperplasia of the interfollicular and deep cortical areas was as well developed as in the preceding forms (Fig. 3). A major plasmocytosis was noted in the medulla (Fig. 3), the cortex, especially the outer areas, and in the germinative centres. One should also note in a few cases with type IA the presence of some follicles with an "onion bulb" aspect.

B

Fig. 3. Case no. 16. ARC type lB. a) Germinal centre with onion skin like aspect. Vascular hyperplasia in the follicle and in the interfollicular zone. Atrophy of the deep cortex. Severe plasmacytosis in the medullary cords. HES. G = 130 x. b) A great number of immunosecreting cells are seen in the germinal centre, in the interfollicular spaces and in the medullary cords. Immunofluorescence on paraffine section, mu heavy chain. G = 200 x.

598 .

J. Diebold et al.

1.3. The lymphadenopathies resembling angioimmunoblastic lymphadenopathy (AIL) or type II In 2 patients (2nd lymph-node biopsy in cases 6 and 10), there existed an aspect which simulated AIL by the destruction of the germinative centres by a large lymphocytic infiltration, the infiltration of the interfollicular zones and deep cortical area by centroblasts and immunoblasts, and vascular hyperplasia (Fig. 4A). However, the persistence of follicular remnants, particularly on silver impregna-

tions, and the existence of various sinusal reactions allowed real AIL to be eliminated. In 7 lymph-nodes of our series taken from 6 patients, an AIL-like aspect was only noted in certain areas, whereas the rest of the lymph-node presented a follicular hyperplasia. Such an association of lesions (type I-II) indicates a possible evolution from a follicular hyperplasia to an aspect like that of AIL. This possibility was confirmed by the histopathological modifications observed when successive lymph-node biopsies were performed. In two cases (no. 6 and 10), the first lymph-node showed a lesion of type IA and the second a type II, associated in the case 10 with a Kaposi's sarcoma. Furthermore, in case 7, the first lymph-node was of type IA whereas the second showed an association of types IA and II.

2. Lymph-node modifications in the confirmed AIDS phase

A

B

Fig. 4. a) Case no. 43. ARC type II. Effacement of the normal structure. No follicle recognizable. Vascular hyperplasia. Polymorphous cell population. HES. G = 125 x. b) Case no. 25. AIDS type III. Lymphoid depletion. Sclerosis destroying the follicles. Vascular hyperplasia. HES. G = 85 x.

This group comprised 16 patients, 13 men and 2 women (Table 2). Ten of the men were homosexuals, and one of these a drug-addict. Moreover, one was of Haitian origin and the other African. The 3 heterosexual nondrug takers were 2 Haitians and 1 man from Zaire. Two of the 3 women were from Haiti, the other from Zaire. The latter had given birth to a child who had been treated ever since for AIDS. Finally, one of the Haitian women was the wife of one of the Haitians (cases 27 and 28). In AIDS, outside the site of an infection or Kaposi sarcoma, the adenopathies disappear. Only four lymph-nodes were studied by biopsy (cases 25, 34, 36, 37). The others were systematically taken during autopsy, in the mesentric and/or mediastinal areas. A total of 18 lymph-nodes were thus studied.

2.1. Lymph-nodes with lymphoid depletion (type III) In our series, 15 small lymph-nodes taken from 14 patients presented this aspect of lymphoid depletion (Fig. 4B). These were always small lymph-nodes less than 1 em long. The lymph-node parenchyma no longer showed any recognizable structure; the follicles had disappeared. A systematized collagen sclerosis thickened the normal framework in the cortex as well as the medulla. Sometimes hyalin deposits indicated the location of the old follicles. Areas of interstitial oedema were visible in places. The cellular depletion involved chiefly the lymphocytes. In contrast, there still remained numerous immunoblasts and plasmocytes. Similarly, histiocytes developing a macrophagic activity, more or less intense, were observed in quite large numbers. Cellular depletion and sclerosis made it easier to visualize the framework and the small vessels which sometimes formed aspects difficult to distinguish from an early lesion of Kaposi sarcoma. In all these cases but one (no. 36), there was no adenopathy. The lymphnodes were small and found with difficulty during necropsy. The only one which was biopsied was a small mesenteric lymph-node taken during a laparotomy in a patient with abdominal infectious disease (case no. 36).

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 599

2.2. Localization of infectious diseases During 4 autopsies, in addition to the lesions already described, lesions were discovered which were due to the pullutation of various infectious agents. In one case (no. 26), the lymph-nodes were partially destroyed by plagues of caseous necrosis, rich in Koch bacillus, without an epithelioid reaction, showing an acute tuberculosis. In another case (no. 38) loci of cryptococcosis were present. In two other cases, the predominant lymph-node lesion was an extensive histiocytosis. This was situated in the sinus and a little in the lymph node parenchyma in case no. 29, with the presence in the cytoplasm of the macrophages of small PAS positive and Gram positive grains, thus presenting an aspect simulating Whipple's disease (Fig. 5 A). In the other case (no. 32) the histiocytosis was more dif-

fuse, infiltrating the lymph-node tissue more extensively. These macrophages contained numerous Ziehl positive rods, evoking an atypical mycobacteriosis which was in fact confirmed on culture which showed mycobacterium aviae (Fig. 5 B).

2.3. Other aspects of lymph-node in patients with AIDS Biopsies were performed on 3 lymph-nodes: one of the patients is now dead (case no. 25) and two still alive (cases 34 and 42). In two cases, the lymph node showed the aspect of diffuse hyperplasia simulating AIL (cases 25 and 42). In the last case (no. 34), aspects of follicular hyperplasia and partial areas simulating AIL were associated. These data plead also in favour of a continuous evolution from the modifications due to hyperplasia seen during ARC to the depletion of confirmed AIDS, according to a hypothetical schema proposed in Table 3. In these 3 cases, the lymph-node biopsy was probably performed at the beginning of the transformation of ARC in AIDS.

3. Lymph-node localizations of Kaposi sarcoma (type IV) Kaposi sarcoma appeared in 10 patients in our series, 3 of whom corresponded to the definition of ARC (cases lOb, 13, 16), whereas the other 7 presented an AIDS (cases 25, 27, 29, 30, 35, 36, 37). The lymph-node involvement is not constant. Only one patient from the first group (case lOb) and 5 from the second group (25 b, 27, 29 a, 30, 35) had lymph-node localization. This was constituted, as in other Kaposi sarcomas, by a proliferation of fusiform cells demarcating vascular spaces containing erythrocytes, accompanied by deposits of collagenous fibres and macrophages with haemosiderin. Lymph node involvement may be massive (type IVA), destroying the entire parenchyma (case 35) or responsible for several tumorous formation which begin in the medullary area (case 30). It can be parcelled, even minimum and discreet (type IVB). In these cases (no. 10 b, 25 b, 27, 29 a) the lesions resided in the lymph-node capsule or in the perivascular septae. Diagnosis can be very difficult. In patients with AIDS and Kaposi sarcoma, the lymph-nodes exhibited lymphoid depletion aspect (type III). In patients with ARC the lymph-node lesions were in one case (no. 13) of the type I and II association, in another (case 10 b) of the pseudo AIL type (type II) and in the last (case 16) of type IB, multicentric Castleman's disease like lesions.

4. Immunohistopathological study after inclusion in paraffine, by direct immunofluorescence

Fig. 5. a) Case no. 29. AIDS. Diffuse histiocytosis with Whipple like lesions. HES. G = 100 x. b) Case no. 32. AIDS. Diffuse histiocytosis due to mycobacterium avium. HES. G = 160 x. Inset: The histiocytes are filled with Ziehl positive germs. Ziehl coloration. G = 580 x.

This study demonstrated only cells with intracytoplasmic immunoglobulins. Plasma cells had excentric cytoplasm marked in an intense and diffuse way or occasionally in the form of multiple little confluent round inclusions. Plasmoblasts exhibited the same aspect but were larger. Centroblasts can be recognized by the existence of a narrow perivascular rim with occasionally positive small intranuclear vacuoles. The presence, in the same area of positive cells for either the kappa or the lambda light chain was considered indicative of a polyclonal population.

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4.1. In 13 lymph nodes with predominant follicular hyperplasia (cases 1, 2, 3, 5, 6 a, 8, 10 a, 15, 18, 19, 21,

22,24), numerous polyclonal immunosecretory cells were found. In all the cases, a polyclonal plasmacytosis was seen in the germinative centres. They were dispersed in the centre or disposed in clusters either in the middle or at the periphery of the germinative centre. This intra follicular plasmacytosis was moderate in 8 cases, severe in 5. In 3 cases (no. 1, 5, 15) the plasma cells secreted only the gamma heavy chain. In one case, they produce only the mu heavy chain (6 a). In the 9 other cases, gamma heavy chain producing plasma cells were predominant but in association with some plasma cells containing in one case (no. 3) the alpha heavy chain, in 5 cases (no. 2,8,10 a, 18) the mu heavy chain, in 3 cases (no. 19,21,22) either the alpha or the mu chain. This intrafollicular plasmacytosis was associated in 11 cases with a variable number of immunosecreting large cells with a centro blast aspect. In 6 cases (no. 5, 6a, 8, lOa, 15,24), those centroblasts produced only the mu heavy chain. In the 5 other cases (no. 2, 3, 19, 21, 22) besides mu secreting centro blasts, some alpha containing centroblasts were discovered. In the interfollicular and medullary zones, a plasmocytosis of variable intensity was noted in the 13 cases studied. Always poly clonal, this comprises a majority of plasma cells secreting the gamma heavy chain associated with a few alpha cells and occasional mu cells. In 9 cases, these plasma cells were associated with large immunosecretory cells that could correspond to centroblasts or immunoblasts with a more or less plasmoblastic differentiation (cases 1, 3, 5, 6 a, 10 a, 19,21,22,24). These large cells comprised in 7 cases cells secreting the mu chain, 3 times alone (cases 19, 21, 22), 3 times associated with alpha cells (cases 3, 6 a, 10 a), once with large gamma cells (case 1). In 2 cases, the large cells contain only the alpha chain. 4.2. Two lymph-nodes presenting a pseudo-AIL diffuse hyperplasia (type II) were studied (cases 6b and lOb). In

both cases, an important polyclonal plasmocytosis was discovered, with a great predominance of gamma plasma cells but always associated with a lower number of alpha and with rare mu plasma cells. Follicular remnants were located on the presence in the external cortex of delimited areas comprising mu centro blasts, either kappa or lambda.

4.3. In the 3 lymph nodes exhibiting an association of type IA and type II lesions (cases 4, 7b, 17), polyclonal

centroblasts were recognized in the germinal centre. In one case, all the centroblasts were mu positive (case 17). In another case (no. 7 b) they all were gamma positive. In the 3rd case (no. 4) some contained mu heavy chain, others gamma chain. An intrafollicular plasmacytosis was associated in 2 cases (no. 4, 7b). In the pseudo-AIL areas a severe polyclonal plasmocytosis was found in all the cases, with predominance of gamma plasma cells, in association with some alpha containing plasma cells and rare mu positive plasma cells. In case no. 4, polyclonal immunoblasts containing either the alpha or the mu heavy chain were found in association with the plasma cells.

4.4. In the only lymph nodes presenting a pseudo-Castleman hyperplasia or type I B (case 16), a considerable

quantity of immunosecretory cells was observed (Fig. 3 B). In the follicles, these are essentially centroblasts with rare plasma cells. These cells contain exclusively the mu heavy chain with such a predominance of the lambda light chain (8 cells per field, per follicle) over the kappa chain (1 positive cell every 4 follicles) that certain follicles seem to contain a monoclonal mu lambda population. In the interfollicular spaces, atrophic paracortex area, as well as in the medullary area, the immunosecretory cells, packed side by side, possess the morphology of plasma cells, and sometimes of immunoblasts with plasmoblastic differentiation. These cells are polyclonal, with a presence of the 3 principal heavy chains, but with a strong predominance of the gamma chain, as demonstrated by the number of positive cells per field (gamma: 50, alpha: 15, mu: 2). 4.5. Only two patients with AIDS were the subject of an immunohistological study. In one case (no. 34), the histological aspect was that of a deep cortical hyperplasia, AIL-like. The study was carried out on an area with no follicular hyperplasia, and demonstrated the existence of an important reactional polyclonal plasmocytosis with no predominance of one heavy chain over the others. In the second case (no. 25), the histological aspect was based on the biopsy of a diffuse hyperplasia simulating an AIL, and on a lymph-node taken during autopsy, with a lymphoid depletion aspect. In both lymph nodes the immunohistological study demonstrated an important polyclonal plasmocytosis with the presence of the 3 heavy chains, but with a predominance of the gamma heavy chain.

5. Detection of histiomonocytic enzymes after inclusion in paraffin by the Sternberger PAP method No particular observation arose from this study, carried out on 4 confirmed AIDS cases (25 a and b, 29 a and b, 34, 36) and on 14 cases of ARC (no. 1-4,6 a, 7, 8, 10 a and b, 16, 18,20-22,24). The study simply confirmed the presence of histiocytes in the sinuses and in the lymph-node parenchyma (germinative centre, juxta sinusal cortical area).

6. Immunohistopathological study on frozen samples of 12 subjects with AR C 6.1. In eight lymph-nodes showing a predominant follicular hyperplasia (cases 7, 9, 18,21,22,24,41,43), the pan-B markers showed an important global B hyperplasia. Furthermore, in all 8 cases, a surface immunoglobulin (S Ig) was shown on most of the centro follicular cells. This comprised the kappa or lambda light chains. In all cases, the majority of the centrofollicular cells were positive with anti-mu serum. In 4 cases (no. 7, 9, 40, 41) there was an association of 10-30% of lymphoid cells in one or more follicles, expressing a S Ig possessing the alpha heavy chain. Three facts concerning the follicles seem to be particularly worthy of attention. Firstly, the lymphocytes of the perifollicular mantle zone presented polyclonal S Ig possessing the heavy mu and delta chain. The anti-delta

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 601

serum clearly demonstrates in all 8 cases that numerous perifollicular mantle zones were depleted and of irregular thickness (Fig. 6B). They are often disorganised, burst, and interrupted. They have occasionally disappeared completely. Furthermore, a variable quantity of delta lympho-

A

Fig. 6. a) Case no. 45. ARC type IA. Immunoperoxidase staining of cryostat section with OKT8 showing increased suppressor cytotoxic lymphocytes within the germinal centre; these OKT8 + cells are either dispersed or in clusters. The number of OKT8 + lymphocytes is also increased in the deep cortex. G = 200 x. No counterstain. b) Case no. 45. ARC type IA. Immunoperoxidase staining of cryostat section with IgD. Delta + follicular mantle cells are decreased and realize a loose and narrow layer, whereas same delta + cells are scattered in the interfollicular areas. G = 200 x. No counterstain.

cytes were to be found in the interfollicular regions and the deep cortical regions (cases 7, 21, 22, 34, 40, 41). They may be dispersed or assembled in small nests. A second, equally important fact, concerns the T lymphocytes presenting a cytotoxic-suppressor phenotype. Thus, in 4 cases (no. 7,22,41,43), an abnormally high number of positive cells with OKT 8 antibody is observed (Fig. 6 A). These may be dispersed, or assembled in clumps of varying magnitude. In most follicles they represent over 10% of the centro follicular lymphocytes. In certain germinative centres they may even reach the figure of 20 to 25% of the cell population. In the other 4 cases, the number of OKT 8 positive cells did not exceed 5 to 10%, and did not seem significantly increased in relation to the control lymphnodes. Nevertheless, the distribution of these cells is abnormal. Instead of being regularly dispersed in the germinative centres, they are assembled in clumps, either central or, occasionally, peripheral (cases 9, 18), continuous with the perifollicular areas. Finally, in the more or less wide sectors made up of positive OKT 8 lymphocytes, the disappearance of the reticular dendritic cells can be observed, easily located using an antibody recognizing receptor C3b (Fig. 7). This localized disappearance of the dendritic cell network seems to us to represent a highly evocative lesion. Finally one should point out the presence, in the follicles of these 8 cases, of an apparently normal number of T lymphocytes labelled by the OKT 4 antibody. These inducer/helper phenotype lymphocytes represented approximately 5 to 10% of the centro follicular lymphocytes, as in the control lymph-nodes. The interfollicular spaces showed an important polyclonal B lymphoid hyperplasia, which may affect between 20 to 30% of cells, according to the case. In these zones, and in the deep cortical areas, T lymphocytes represent 50 to 80% of the cells, according to the case and to the area. The ratio of T 4/T8 lymphocytes appears to be well below 1 (0.5 on average) in 5 cases (no. 7,18,21,24 and 43). On

Fig. 7. Case no. 22. ARC type 1A. Immunofluorescence staining of consecutive cryostat sections. Section stained for RC 3b : the diffuse network of dendritic reticulum cells contained some negative zone (*), often in continuity with interfollicular areas. G = 330 X. Inset: Section stained for OKT8. Presence of a OKT8 + lymphocytes cluster, in the follicular area. G = 330 x.

C Fig. 8. Case no. 18. ARC. a) Interfollicular area with immunoblast (Ib) transformed lymphocytes (L) and plasma cells (PI). IRS in the cytoplasm of a transformed lymophocyte (J'). G = 4,900 x. b) Small lymphocyte with IRS (J') in the perinuclear cisternae. G = 20,000 x. c) Detail of the IRS. G = 80,000 x.

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 603

the other hand the ratio was very close to 1 in 3 cases (no. 9,22 and 41). But there is a very heterogenous distribution of the T sub-populations from one area to another. At places, the T 4 lymphocytes existed in quantities closely resembling those observed in the control lymph nodes. There are very few elsewhere. This heterogenous distribution rendered an exact evaluation of the T4/T8 relationship in the lymph-node very difficult. On the whole, however, the quantity of T8 lymphocytes appears to be superior to that observed in the control lymph-nodes, there being no correlation between their number in the germinative centres and outside the follicles. The quantity of T8 lymphocytes is thus considerable, in the 4 cases where these cells are numerous in the follicles (cases 7, 22, 41, 43), and represents 50 to 70% of the extrafollicular zone lymphocytes. T8 lymphocytes are also numerous outside the follicles in the 2 cases (no. 18 and 21) where the number of TS lymphocytes in the germinative centres is not considerably high. Furthermore, an abnormally high number of interdigitating cells, stained by the OKT6 antibody, can be noted in the deep cortical areas. This hyperplasia may remain moderate (cases 9, 21, 40), or be severe, capable of reaching 15-25 interdigitating cells per field at a magnification of 200 x. In these areas, the vascular hyperplasia predominant on the epithelioid venules was well demonstrated by the monoclonal ALB6 antibody. Finally, a variable quantity of large cells stained with OKMI antibody can be recognized in the sinuses, particularly in the peripherals, which corresponds to a sinusal histiocytosis. In three cases (no. 24, 41, 43), this is associated with a number of cells stained with OKT6 antibody in the subcapsular sinus. 6.2. In the unique case with type II histological aspect (no. 42), the T lymphocytes represent 30 to 50% of cells, according to the area, with an almost equal quantity of T 4 and T8 cells, and thus a proportion differing little from 1. However, these two types of T lymphocyte are distributed in a highly heterogenous manner according to the area. They are associated with an important lymphoid B hyperplasia, representing approximately 40% of the cells. These cells possess a S Ig with one or the other of the light chains, and a distinct predominance of the heavy mu chain, but no C3b receptors. A considerable number of cells stained with OKT6 antibody, probably interdigitating cells, can be detected in areas rich in T-lymphocytes, up to 15 cells per field for a magnification of 200 x, which enables the location of the deep cortical areas. The ALB6 antibody emphasised the vascular hyperplasia in these same zones. Furthermore, this study revealed follicular remnants dispersed in the lymph-node. These appeared as oval or rounded zones consisting of cells possessing the C3b receptor and as S Ig with a mu kappa or mu lambda predominance occasionally with 10 to 20% alpha containing cells. Again, incomplete perifollicular mantle zone of varying thickness can be recognized by revealing the delta S Ig. In the peripheral, and occasionally interfollicular sinuses, histiocytic cells stained with OKMI antibody, in addition to a few OKT6 positive cells, were recognized.

6.3. In two cases (no. 34 and 40), the histological aspect associates follicular hyperplasia predominant areas with others simulating an AIL. The observations from immunolabelling on frozen samples superimposed on the preceeding cases. The only difference consists in a distinct follicular hyperplasia with a lymphoid population of the type observed in cases with predominant follicular hyperplasia.

7. Ultrastructural aspects 7.1. The electron microscopic study of 11 patients with ARC or AIDS showed many cytoplasmic particularities. 7.1.1. Two major types of cytoplasmic inclusions were seen: tubuloreticular structures (TRS) and test tube and ring shaped forms (TRF). TRS were observed in the 9 patients with ARC and in the 2 with AIDS. They consisted of anastomosing tubular structures of less than 30 nm in diameter (Fig. 8A and B). They were usually located in the endoplasmic reticulum (ER) but, frequently, the ER membranes were not clearly visible. Sometimes the TRS were present within the perinuclear cisterna. The cells involved were endothelial cells and lymphocytes. TRF were observed in 2 cases with ARC (cases no. 7 and 16) and in the 2 cases with confirmed AIDS (cases no. 34 and 36). These structures were only located in the lymphocytes. They were usually composed of two concentrically ER cisternae. They were ring shaped when horizontally sectionned and tube shaped when longitudinally sectionned. Some electron dense cytoplasmic material was placed between the inner membrane of each ER cisternae and seemed to cause them to adhere together (Fig. 91A and B). 7.1.2. Other types of cytoplasmic inclusions must be noted or discussed. Vesicular rosettes (VR) were not seen clearly in any case in our study. Some clusters of vesicles were sometimes present but never placed around an electron dense core as in Ewing's description21 • They were interpretated as some sections of the Golgi area. Numerous multivesicular bodies (MVB) were seen in the endothelial cells and in some lymphocytes and macrophages in all

Fig. 9. Case no. 7. ARC. TRF in the cytoplasm of a lymphocyte. G = 22,000 x.

A

B

Fig. 10. Case no. 21 b. ARC. a) Germinative centre with centroblast (Cb), plasma cells (PI), and dendritic reticular cell cytoplasm realizing a labyrinth (*) with intercellular junctions (1'). G = 4,800 x . b) Labyrinthic organization of the dendritic reticular cell cytoplasmic prolongements with cell junction (1'1') and some virus like particle. G = 15,200 x. c) Virus like particles looking like D particles can be recognized between altered cytoplasmic projections of dendritic reticular cells. G = 30,000 x.

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 605

our cases. In 5 cases of ARC (cases 2,9, 16,22,24), some cisternae of the cytoplasmic membrane system had fusion of the paired membranes analogous to the so-called annulate lamellae. In 4 cases, 3 with ARC (cases 16,21 b, 24), and 1 with AIDS (case 34), helicoidal polyribosomes were present in the cytoplasm of the lymphocytes and endothelial cells.

7.2. In the 9 patients with ARC, areas with hyperplastic follicles were found after cutting all blocks only in 7 cases (case 2, 7, 18, 21 b, 22, 24, 41). In all the patients with such follicular hyperplasia, between the centrocytes, centroblasts and tingible body macrophages, some reticular dendritic cells were found (Fig. lOA). Their cytoplasmic extensions composed a distinctive labyrinthine complex. Between the villi form cytoplasmic projections, interlacing with those of adjacent cells, prominent desmosomal junctions could be recognized (Fig. lOB). In the extra cellular spaces of this labyrinthine complex, numerous virus like particles were dispersed (Fig. 10C). Most of the particles looked more or less like C on D particles characteristic of RNA retrovirus 6. They had a distinct limiting membrane and a central or eccentric dense core. These particles look very alike those described by Armstrong and Horne3in the same site in 6 patients with ARC. We were not able to recognize the particles budding from membranes of the expanded dendritic reticular cell processes. No particles were seen in the cytoplasm of dendritic reticular cells, centrocytes, centroblasts, plasma cells or macrophages. 7.3. In the 2 cases with massive histiocytosis, the lymphnodes were badly preserved, due to the initial Bouin's fixation. It was thus impossible to observe any type of previously described inclusion. Nevertheless, sections of bacterial corpuscules could be recognized in both cases. In case no. 29, at the optical microscopic level, the histiocytosis looked like Whipple's disease. At the ultrastructural level, some bacterial corpuscles were recognized in the cytoplasm of all the macrophages, in association with numer-

Fig. 11. Case no. 32. AIDS. Diffuse histiocytosis. A high number of mycobacteries are seen in the cytoplasm of a macrophage without any picture of digestion. Lymph-node fixed with Bouin's solution. G ::= 8,000 x.

ous phagosomes of various size and morphology. In case no. 32, the aspect was that of a mycobacterial histiocytosis. At the ultrastructural level, bacteria were detected not only in the cytoplasm of macrophages but also between the cells. They were not associated with phagosomes and it was not possible to recognize any aspect of intracellular digestion (Fig. 11). Discussion In confirmed AIDS, the lymph-nodes are mostly atrophic (77% of our series), corresponding to our histological type III. This atrophy is due to a lymphoid depletion with disappearance of the follicles, plasmocytosis, histiocytic hyperplasia, oedema and systematic scleroSiS 7,17,28,44,t.l,54,65. The few cases studied by immunofluorescence showed the persistance of a B lymphoid hyperplasia with numerous polyclonal immunosecreting cells, which suggests that the lymphoid depletion could predominantly involve the T cells. Unfortunately, the absence of frozen samples from this type of patient did not allow the T lymphocytes to be studied using immunolabelling. When adenopathy still exists, this is due either to localisations of various infectious diseases 4,33, 58, 59, 72 or to the presence of a Kaposi sarcoma 10,20,30,42,43,46,58,59, 69,72. Concerning the infectious diseases, in our series this was usually acute tuberculosis, cryptococcosis, or atypical mycobacteriosis58, 59. One observation (no. 29), from which the infectious agent could not be isolated, presented a histological aspect recalling Whipple's disease. The ultrastructural study allowed signs of digestion associated with intracytoplasmic bacteria to be noted in the macrophages, whereas in the case of infection with mycobacterian avium, no phagosome could be found. These facts probably reflect different dysfunctional states of the macrophages. The lymph-node sites of Kaposi sarcoma ressemble the disseminated lymphoid forms already described, in particular in young people, especially in Africa7, 19,23,56,'74,80,81. In the lymph-nodes, the two aspects described in disseminated Kaposi sarcoma can both be recognized7 : a massive, often medullar attack or a localised peripheral involvement. Such partial localizations occuring in the capsule or the spetae would seem to be rather specific to AIDS30. They can be very difficult to recognize. The detection of factor VIII, by immunolabelling, in tumoral fusiform cells could perhaps be useful for diagnosis32. The development of a Kaposi's sarcoma can be explained by different hypothesis which can associate 10 : proliferation of endothelial cells after transformation by a Herpes-type virus; production by the lymphocytes of a factor stimulating angiogenesis 55 ; tumor development secondary to a T lymphoid deficiency with a decrease in NK cell activity. This last mechanism is also evoked to explain the appearance of malignant lymphomas which are often outside the lymph-node, apparently of B type and of high malignancy1!, 48, 69, 85, 86. The T lymphoid deficiency installs progressively, often during the course of ARC, to finally become complete and worsened in AIDS.

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The patient with an ARC appears more interesting to AIL. Such an aspect was also noted subsequently in other study. The lymph-node hyperplasia is due in all cases to a series 8, 28, 50, 72. A different diagnosis is usually possible major B lymphoid hyperplasia associated with a histiocytic due to follicular remnants which are more developed than hyperplasia, and a variable hyperplasia of the thy- in real AIL and which are particularly well visible in immodependant areas with numerous epithelioid venules. munohistopathology. The histological modifications allow these adenopathies to Intermediate forms (20% of the cases in our series) be divided into 3 groups of different histological aspect. associating, in the same lymph-node, a follicular hyperThe forms with predominant follicular hyperplasia or plasia in certain areas and a pseudo-AIL hyperplasia of the rr,l'e lA, are the most frequent 9,20, 22, 28, 2~, 43, 49, 50, 58, 59, deep cortical area with disappearance of the follicles in 7 ,87. This picture was seen in 71 % of the lymph-nodes of others, suggest a possible evolution from one form (type I) our cases. The deep cortex can be more or less hyperplas- to the other (type II) (Table 3). tic, as in our cases, but the vascular hyperplasia is consisIn our patients fulfilling the definition of ARC, 5 (17%) tanr9,20, 58, 59, 87. Sometimes, small areas of lymphoid evolved to a confirmed AIDS. Two patients showed depletion with oedema and/or sclerosis can be noted9, 20, lymph-node lesions of type IA (cases 8, 11), another type 49, 50, 58, 59, 87. The histiocytic hyperplasia can take the I-II associated (case 13), and another (case 10) showed on form of various types of sinusal reaction with cell successive biopsy a change from type I to type II. The last phagocytosis, especially erythro~hagoc;;tosis as well as by case (no. 16) was the only one with multicentric Castlethe development of histiocytic' 20, 58, 9,87 or epithelioid man-like lymphadenopathy (type IB) in our series. It could granulomas 20,58, 59. Such histiocytic hyperplasia impose thus be feared that histiological types IB and II evolve systematically special staining procedures (PAS, Grocott, towards an AIDS. The same is true for lymph-node lesions Gram, Ziehl) which allow various infectious agents to be associating type IA and II, due to a progressive destruction detected. Various types of sinus reaction can be found, in of some germinative centres by a lymphoid infiltration of particular sinusal B lymphocytosis called in the past cells with a cytotoxic/suppressor phenotype (T8). The sinusal immature histiocytosis. Sometimes a hYferplasia of existence of small loci of lymphoid depletion due to plasmocytoid T lymphocytes can also be seen 3. The his- sclerosis or oedema also perhaps represent signs of a tiological aspects of these forms with predominant follicu- danger of evolution towards an AIDS although this evenlar hyperplasia are not specific to an ARC. Other tuality was not noted in any of our cases. But it is etiologiesrnustbe considered, in particular toxoplasmosis, nevertheless difficult during an ARC to find sufficient evidiverse viral infections or even syphilis. Nevertheless, sev- dence to predict evolution to AIDS 52,57. eral histiological modification, although not specific, The retrospective study of our patients, having conattract attention. These are essentially the disappearance firmed AIDS, allowed the existence of an ARC to be cliniof the perifollicular lymphocytic mantle and the aspects of cally and biologically proven in 9 cases out of 16 (56%). It seems interesting to stress that of the four biopsied infiltration of the germinal centres by streams of small lymphocytes. The interest of these histopathological signs lymph-nodes of patients having confirmed AIDS in our is underlined by the immunochemical data: disappearance series, one had the aspect of an association of types I and from the perifollicular mantle zone of lymphocytes posses- - II, and 2 that of the type II. These observations link the sing membrane IgD with migration of these lymphocytes groups of patients with ARC or with AIDS and suggest the to the deep cortex, disruption of the network of dendritic existence of a common factor 2o, 29, 58. Furthermore, several reticular cells, and infiltration of the germinal centres by a cases with successive biopsies show a transformation of higher number than normal of lymphocytes having a the histological aspects, on the one hand of type I to that phenotype of cytotoxic/suppressor cells (8) and being of type II, and on the other hand of type I-II and type II to responsible for the disruption of the dendritic reticular cell that of type III. Such observations suggest that there are definite links and continuous evolution. Other arguments, network. A particular form (ty~e IB) simulating multicentric in particular epidemiolo~ical, have also been reported in Castleman syndrome34,3 or angiofollicular poly- favour of this hypothesis 3. It is thus possible to propose as adenopathies with plasmacytosis 18 have also been a working hypothesis, to be verified by prolonged followobserved. This form is still rare20, 58, 59. In our only case, a up, an evolutive picture or schema of the histopathological cutaneous Kaposi sarcoma occured in the weeks following aspects (Table 3). The primary lesion would be a predothe lymph-node biopsy, with a rapidly fatal evolution. One minant follicular hyperplasia. Then aspects of focal points should remind the publications in the past of such an of pseudo-AIL would appear, which would progressively association in patients without known risk factors for take the place of all areas of the lymph-node. Then the AIDS 56, 74. Similar observations have been lymphoid atrophy would worsen, allowing immunoblasts reported28, 29, 46, 69 since the publication of our case20, 58, 59. to persist, as well as plasmocytes and histiocytes. At the One of these69 also included a lymphoplasmatic ML, mu same time an intercellular systematic sclerosis would lambda. In our case, immunolabeling technics detected a develop. At this stage, the adenopathy would disappear. mu lambda B cell population in the follicles. This popula- Various infectious lesions or Kaposi's sarcoma could then tion seems to be practically monoclonal. It could corres- develop in the lymph-nodes. In our series, no malignant lymphoma could be found, pond to very early beginning of a clonal proliferation. The last 7pe, or type II, also mentioned in our first whereas in various different publications, several cases of publicationS ,has an aspect more or less close to that of an B cells ML have been reported ll , 12, 21, 48, 60, 72, 84-86.

Lymph Node Modifications in Patients with AIDS or with AIDS Related Complex . 607 Table 3. Diagram of the Possible Evolution of Lymph Node Lesions in LAS and AIDS Type I A. Lymphoid follicular hyperplasia

Type I B. Multicentric Castleman-like lymphadenopathy

Association of types I and II Type II AIL-like lesion

Type IV A or B Kaposi's sarcoma

Type III Lymphoid depletion Resolution (?)

The immunohistopathology on frozen sections using monoclonal antibodies detects several facts useful for the diagnosis of ARC. The value of these modifications could be evalued by comparison with the data obtained on stimulated lymph-nodes of different etiology. The number of T lymphocytes in the follicles is identical to that observed in other pathological states, or more often even higher. The most important fact is that most have a suppressor/cytotoxic (TS +) phenotype, whereas the number of the helper/inducers (T 4 +) is reduced or remains normal. Similarly, in the interfollicular and deep cortical areas there is an increase of TS lymphocytes, with a lower or equal number of T 4 lymphocytes as in the control lymphnodes31 , 61, 64, 65, 75. The inversion of the T4/T8 ratio is often difficult to appreciate due to the great heterogenicity in cell type from one lymph-node to another. However, as well as the inversion of this ratio, the distribution of the TS lymphocytes, with an important infiltration of the germinative centres, also seems to us to be an excellent sign for suspicion. The correlation with the lowering of the ratio of T lymphocytes in peripheral blood is nevertheless difficult to establish. Of the 10 patients of the ARC group having a clearly lower ratio, only 2 had an immunolabelling study performed on the lymph-nodes. In case 7, the T4/T8 ratio was very reduced in the lymph-node. In case 9, the ratio was normal, but the distribution of TS lymphocytes was abnormal, forming an infiltration in the form of a band, dissecting the follicles. This aspect of lymphocyte infiltrating of the germinative centres evolving towards dislocation, or bursting of the follicles, is an excellent sign which evokes the diagnosis. This destructive character of the TS lymphocytes in the follicle is best demonstrated by the detection of dendritic reticular cells either by an antibody labelling the C3b receptors or by a monoclonal antibody specific for this cell type (KiM4 produced by Dr. Feller, Dr. Radzun and Prof. Lennert). Furthermore, the immunolabeling studies demonstrated an important polyclonal B hyperplasia without any special

particularity. In type IA lymph nodes, there is a heavy predominance of large intrafollicular immunosecreting cells of centroblastic type, containing polyclonal IgM, while the intra- and extra-follicular plasmocytosis, also poly clonal, is, predominantly IgG. Such aspects are observed in all B hyperplasias, regardless of the etiology. The polyclonal IgG plasmocytes predominate in type II, whilst the nests of centroblasts predominantly IgM, allow the traces of the burst follicles to be found. In type III, an IgG predominant B hyperplasia persists. However, the most interesting factor for diagnosis is represented in type I and II by the progressive disappearance of the perifollicular mantle zone, well demonstrated by the study of membrane IgD. The delta lymphocytes disappear from the perifollicular mantle zone and migrate to the deep cortical and interfollicular areas 60, 61, 64, 79. A hyperplasia of the histiomonocytes was also noticed and well-confirmed by the detection of various enzymes using immunoperoxidase, or of certain antigens using monoclonal antibodies. This hyperplasia is observed in all the described histological types. It should be compared in types I and II to that of interdigitating cells in the deep cortical area and, in certain cases, the presence of these cells in the sinus. At the ultrastructural level, the 2 most important features are represented by the discovery of virus like particles in the hyperplastic germinal centre of patients with ARC and the detection of TRF in some lymphocytes in patients with ARC and AIDS. The morphology of the virus-like particles was that of type C or 0 like particles res sembling to retro-virus 3, 6. This discovery was not easy to understand. It could be that virus arriving from extranodal areas, are trapped as an antigen or an immune complex between the dendritic reticular cells, which are called "antigen-trapping" cells67 • Another possibility is that the virus are produced by infected lymphoid cells (activated T 4 cells?) within the lymph-node and concentrated in the dendritic reticular cell network. Such entrapment of virus in the labyrinthine complex made by those cells was described in mice infected with Rauscher and Abelson leukaemia viruses 45 • It was, so far as we know, never described in before the report of Armstrong and Horne3 and ours. Despite the resemblance of those viral particles with retrovirus and particularly with retrovirus discovered in reripheral blood lymphocytes from patients with AIDS 27• 3 .70, it is impossible to say if the viral particles correspond to the etiological agent or to an agent responsible of an intercurrent infection. The discovery of type C or D like viral particles in the patient with ARC support a role for routine electron microscopy in lymph-node biopsy, as an aid to diagnostic of the disease. The detection of TRF in 2 cases of AIDS (no. 34 and 36) and in 2 cases with ARC exhibiting evolution to AIDS, one to a confirmed AIDS (no. 7) and the other presenting only shortly after biopsy a Kaposi's sarcoma (no. 16) seemed also to have some interest. TRF in AIDS was first reported by Sidhu et al. 79 and afterwards by others 68 • 76 • The relation between TRF and AIDS is particular and has no acceptable explanation. Despite the fact that TRF cannot

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be regarded as unique to AIDS or ARC, this type of inclusion may prove to be a useful aid for diagnosis, as was stressed by Ewing et aU4,25. TRF has been described in other conditions. They were first described in the ER of hepatocytes from chimpanzees inoculated with J,lasma of patients with non A non B (NANB) hepatitis ,78. They were also observed in other circumstances: in the leukemic cells in one case of adult T-cellleukemia77, in the lymphoid cells and macrophages in one case of chronic multiple sclerosis 71 , in endothelial cells and lymphocytes in fibrosing alveoli tis associated with vascular collagen disease44 • Among the other ultrastructural aspects published24, n, 38, 53, 68, 76,79, 88, we frequently found a lot of TRS in lymphocytes and endothelial cells in all cases of AIDS and ARC. According to recent papers, TRS are found in AIDS in the cytoplasm of T-Iymphocytes4o,41. Some authors 82 described these TRS as "virus-like particles". As others39, 47, 73, we think that the TRS represents a particular modification of the ER involved in some process of synthesis and present in a great number of pathological circumstances. Recent publication4o,41 emphasize the role of interferon in the formation of TRS in lymphocytes. Duration or intensity of exposure to endogenous interferon may be critical for the formation of TRS in the lymphocyte immune reaction. Virus infection may stimulate interferon production by leucocyte and thus, formation of TRS. As Sidhu et aF9, we did not observe in our cases, the rosette inclusions described by Ewing et a1,24. Another inclusion, the MVB, appears as a frequent metabolic inclusion without any relation with AIDS and ARC or with any other precise disease68 . However, we did observe, both in the AIDS and ARC groups, two particular cytoplasmic structures: annulate lamellae and helical polyribosomes. These structures are not "markers" but are frequent in AIDS and ARC as in other stimulated lymph-nodes. They are probably related to the activation of non exportable protein synthesis. Concerning the two cases with histiocytosis and the presence of intracytoplasmic bacterial particles, the poor quality of the samples did not allow any precise study of bacterial digestion nor whether the bacteria were identical in the two cases. Different clinical, epidemiological and biological arguments seem to plead in favour of a viral etiology of AIDS. From the histopathological viewpoint, 2 groups of lesions can be considered as indirect evidence for a virosis. On one hand, there is the presence of Whartin-Finkeldey type cells, which were initially described in viral diseases, even though these cells can be found in various pathological states without an apparent link to a viral infection 16. On the other, there is the presence of juxtasinusal histiocytic granulomas associated with sinusal histiocytosis with erythrophagocytosis which are also non specific lesions but frequently encountered in viral diseases. Other arguments are the increased number of B lymphoid cells with a decrease T4/T8 ratio and the presence of virus particles in the germinal centres. The virus responsible seems to be a retrovirus related to that implicated in the mali~ant T-Ieukemias-Iymphomas of Japan and the Cariban ,36. This virus called LAV5 or

HIL III36,37 penetrates selectively the T4 lymphoid cells, bringing about a T lymphoid deficiency with reduction in lymphokine, gamma interferon66 and interleukin-2 production13. This T4 population include not only helper cells but also some suppressor cells, some suppressor inducer cells and some cytotoxic cells. The association of these functional disorders is responsible for any opportunistic infections. Also linked to them is a B hyperplasia, responsible for adenopathies and serum polyclonal hypergammopathy54. The mechanism of this B hyperplasia is still unknown. It could be due to a direct stimulation, for example by a virus 54 . The increase in the number of suppressor lymphocytes is perhaps an attempt to block this B hyperplasia. To support this relationship with a retrovirus, it should be noted that 10 of the 14 patients of the ARC group in whom serology to LAV was assayed, were found positive (Table 1). In two of these patients, HTLV III research was also positive (cases 5 and 10). In the group of patients having a confirmed AIDS, the test for LAV was carried out 14 times and found positive 9 times in 8 patients. In the same group, out of 7 tests for HILV III, the result was positive 6 times. Positive results for both assays were found for 4 patients (cases 25,27, 36, 39). In conclusion, in young patients with chronic polyadenopathy, a lymph-node biopsy should always be done. Despite the fact that the histopathological lesions are not specific for ARC, it is possible to suspect the diagnosis, because of the presence of an association of some type of modifications, particularly those affecting the follicles. Immunotyping studies showing modification of the number and repartition of T 4 and T8 lymphocytes bring more argument. Finally the discovery of viral particles of C or D type like in the germinal centres is today for us the best morphological argument in favor of an ARC.

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Received February 5, 1985 . Accepted in revised form March 15, 1985

Key Words: Acquired immune deficiency syndrome (AIDS) - AIDS related complex (ARC) -Immunopathology - Virus Particle - Ultrastructure - Kaposi Sarcoma - Type C viral particle Professor Dr. J. Diebold Service Central "J. Delarue" d'Anatomie et de Cytologie Pathologiques (Prof. Paris, 1 place du Parvis Notre-Dame, 75181 Paris Cedex 04

J.

Diebold). H6tel-Dieu de