Chronic parasitisation mollicute-like · Chronic orbital inflammatory disease: parasitisation oforbitalleucocytesbymollicute-likeorganisms EMIL WIROSTKO,' LEWIS JOHNSON,2 AND BARBARAWIROSTKO1

British Journal of Ophthalmology, 1989, 73, 865-870

Chronic orbital inflammatory disease: parasitisationof orbital leucocytes by mollicute-like organismsEMIL WIROSTKO,' LEWIS JOHNSON,2 AND BARBARA WIROSTKO1

From the 'Edward S Harkness Eye Institute and the 2Department ofPathology, Columbia-Presbyterian MedicalCenter, New York, and the 'College of Physicians and Surgeons, Columbia University, New York, USA

SUMMARY Chronic orbital inflammatory disease (COID) is usually considered non-infectious andidiopathic. Treatment is empirical, palliative, and may not prevent disease progression. COIDoccurs in isolation or in association with various systemic diseases. Exophthalmos may be animportant presenting sign. Vasculitis, lymphoid infiltrates, and granulomas are common.

Mollicute-like organisms (MLO) parasitising and destroying vitreous leucocytes are often found tocause human chronic uveitis when an appropriate search is made. Inoculation of these MLO intomouse eyelids produced chronic uveitis and exophthalmic orbital inflammatory disease. Mollicutesare cell wall deficient bacteria. Extracellular mollicutes cause human and animal diseasescharacterised by lymphoid infiltrates, immunosuppresssion, and autoantibody production.Intracellular morphologically similar bacteria are non-cultivable pathogens termed MLO. Identi-fication is based on direct detection in diseased cells by transmission electron microscopy. MLO are

cytopathogenic and detection is aided by the alterations they produce. MLO replace thecytoplasm, destroy the organelles, and alter the nucleus. This results in cell proliferation,destruction, and dysfunction. MLO parasitise lymphocytes, monocytes, and polymorphonuclearleucocytes. This report describes orbital leucocytes parasitised by MLO in three patients withisolated COID. Rifampicin treatment of MLO disease is discussed.

Chronic orbital inflammatory disease (COID) isusually considered to be non-infectious and idio-pathic.' It occurs in isolation or in association withvarious systemic diseases. ' Exophthalmos may be animportant presenting sign.' Vasculitis, lymphoidinfiltrates, and granuloma formation are commonpathological features.' Mollicute-like organisms(MLO) parasitising and destroying vitreous leuco-cytes are often found to cause chronic uveitis inpatients when an appropriate search is made.2'Inoculation of these MLO into mouse eyelidsproduced chronic uveitis7 and exophthalmic orbitalinflammatory disease.8 MLO became disseminatedto produce lethal chronic inflammatory disease in allorgans.2-"' MLO-parasitised leucocytes were detectedin all the diseased organs.2-"' The disease was progres-sive and similar in all the organs.2-" The initial lesionwas a vasculitis.'-"' This led to tissue lysis, lymphoidinfiltrates, and granuloma formation.'-"'Correspondence to Emil Wirostko, MD, Edward S Harkness EyeInstitute, Columbia-Presbyterian medical Center, 635 West 165thStreet, New York, NY 10032, USA.

Mollicutes are cell-wall-deficient bacteria." Theycan be overlooked or confused with viruses.'21 Theirultrastructural appearance'4 is characteristic. Extra-cellular mollicutes are fastidious,'5 lipid-rich,'6 andcontain a variety of potent cytotoxic substancesincluding nucleases.'2 Extracellular mollicutes causehuman and animal diseases characterised by lymphoidinfiltrates, immunosuppression, and autoantibodyproduction. '7-2"Some morphologically similar organisms are intra-

cellular pathogens.2 '1224 Despite considerableefforts at cultivation by many investigators theconsensus is that these mollicute forms are non-cultivable.24 12122 Their taxonomy is uncertain andthey are termed MLO.'3 Identification of MLO isbased on direct detection within parasitised cells bytransmission electron microscopy.' 13 21-24 MLO arecytopathogenic and their detection is aided by theultrastructural alterations they produce. They replacethe cytoplasm and destroy the organelles,21221 andthey also alter the nucleus, probably as a result ofnuclear parasitisation22 and release of nuclease.24

865

on May 3, 2020 by guest. P

rotected by copyright.http://bjo.bm

j.com/

Br J O

phthalmol: first published as 10.1136/bjo.73.11.865 on 1 N

ovember 1989. D

ownloaded from

http://bjo.bmj.com/

Emil Wirostko, Lewis Johnson, and Barbara Wirostko

This results in cell proliferation, destruction, anddysfunction.' A common and important feature ofMLO disease is vascular involvement. "'MLO parasitise lymphocytes,4 monocytes,5 and

polymorphonuclear leucocytes. They appear asundulating filaments of 0-005-0-01 [m diameter, andpleomorphic trilaminar membrane-bound 0-01-1-0[tm tubulospherical bodies. Some forms develop intospore-like 0 5-07 [m cocci by deposition of electrondense material within the trilaminar membrane. Thecocci have distinctive ultrastructural features. Thefollowing report describes the results of a search forMLO parasitised leucocytes in the orbital tissues ofpatients with isolated COID.

Materials and methods

Three patients with COID served as sources of thestudy materials. None had a history of thyroid,bowel, or other systemic disease. On physical examin-ation all had normal signs and no evidence ofperipheral lymphadenopathy. The results of routinestudies of urine, blood cells and chemistry, andthyroid function were within normal limits in allthree, and the chest roentgenogram of each wasnormal. The COID of each patient was considered tobe non-infectious.' Microbiological studies were notconducted on the excised orbital tissues.

Patient I was a 17-year-old female with a two-yearhistory of progressive left upper eyelid ptosis. Orbitalcomputed axial tomograms were normal. Two

Fig. I Case 1. Chronic 'asculitisinvolving arterioles, capillaries, and 'adventitia ofsmall arteries.(Haematoxylin-eosin, xllO).

separate attempts at surgical correction were initiallysuccessful, but there was gradual return of the ptosis.During a third corrective procedure abnormal orbitaltissue was noted and excised.

Patient 2 was a 61-year-old female with a six-yearhistory of progressive exophthalmos limited to theright orbit. Computed axial tomograms performed atthe onset of the disease showed right orbital patchynon-confluent radiodensities and normal extraocularmuscles. The left orbit was normal. Biopsy thenshowed chronic non-specific inflammation. Treat-ment at that time consisted of combination radiationand chemotherapy. This arrested the disease, but fiveyears later progressive exophthalmos recurred. Arepeated computed axial tomography displayed pro-gression of the right orbital disease. The contralateralorbit was again normal. A biopsy of the diseasedtissue was taken, and that material was studied in thisreport.

Patient 3 was a 65-year-old female with a five-month history of progressive exophthalmos of theright orbit. Computed axial tomograms showedpatchy right orbital non-confluent radiodensities andnormal extraocular muscles. The contralateral orbitwas normal. An orbital biopsy was performed.A portion of each patient's specimen was fixed in

10% formalin, processed for routine paraffin-embedded histological sections, and stained withhaematoxylin-eosin. Another portion of eachpatient's specimen was finely minced, fixed in 150mM cacodylate buffered 2% glutaraldehyde, post-

866



j.com/

Br J O


ovember 1989. D

ownloaded from

http://bjo.bmj.com/

Chronic orbital inflammatory disease: parasitisation oforbital leucocytes by mollicute-like organisms

fixed in osmium tetroxide, and embedded in Araldite(R). All these materials were available for investiga-tional purposes through an Institutional ReviewBoard exempt protocol. The sections stained withhaematoxylin-eosin were studied for COID diag-nostic concurrence under a light microscope. Thicksections embedded in Araldite (R) and stained withtoluidine blue were also studied for leucocyte-richfoci under a light microscope. Multiple ultrathinsections were prepared from such foci and stainedwith uranyl acetate-lead citrate. All available leuco-cytes in these sections were studied for leucocytealterations and MLO parasitisation by a transmissionelectron microscope.

Results

HISTOLOGICAL STUDIESThe numbers of leucocytes varied on each patient'sspecimen (Figs. 1-3). Lymphocytes were the pre-dominant leucocyte in each specimen, but a fewscattered monocytes and polymorphonuclear leuco-cytes were also present. Vasculitis was detected ineach specimen, but it was most evident in thespecimen from patient I (Fig. 1). Each specimen alsoshowed scattered minute lytic foci in the extra-vascular adipose tissue. The specimen from patient 2showed an intense diffuse lymphoid infiltrate (Fig.2). The specimen from patient 3 showed several non-necrotic, non-confluent, epithelioid granulomasinterspersed between focally intense lymphoidinfiltrates (Fig. 3). Ziehl-Nielsen, Gomori's metha-

Fig. 2 Case 2. A diffuse lymphocytic infiltrate; an inflamedarteriole is also detectable. (Haematoxylin-eosin, x 155).

Fig. 3 Case 3. Non-necrotic epithelioid granuloma, alymphocytic infiltrate, andfibrosis. No acid fast bacilli orfungi were detected in this specimen. (Haematoxylin-eosin,x 100).

namine silver, and periodic acid Schiff stains on thisspecimen were negative for acid fast bacilli and fungi.

TRANSMISSION ELECTRON MICROSCOPIC STUDIESAbout 2-10% of all the leucocytes in each patient'sspecimen displayed intracytoplasmic undulating fila-ments, 0-005-001 [tm diameter, and pleomorphictrilaminar membrane-bound 0 01-1-0 [tm tubulo-spherical bodies (Figs. 4,5). Some of these bodiesexhibited deposition of intramembranous electrondense material to form 0-5-0-7 [m cocci (Fig. 5). Allthese cocci had thick cell walls, a distinct periplasm,and complex internal tubulofilamentous substruc-tures. Nuclei of leucocytes harbouring the abnormalbodies often had ragged, irregular contours,chromatin clumping with lysis, and prominent peri-nuclear electron lucency (Figs. 4,5). In the poly-morphonuclear leucocytes these bodies were usuallyassociated with cytoskeletal lysis and a paucity ofnormal granules (Fig. 5).

Discussion

This study shows the diagnostic value of examiningleucocyte COID lesions by transmission electronmicroscopy. Clinically and pathologically eachpatient's disease was considered to be non-infectiousand idiopathic.' However, the leucocytes in eachspecimen contained intracytoplasmic bacteria-like bodies. Most of these lacked a cell wall. Some

867



j.com/

Br J O


ovember 1989. D

ownloaded from

http://bjo.bmj.com/


Fig. 4 An aggregate of undulatingtubules replaces the cytoplasm onthe right. The remaining cytoplasmappears normal. A prominentflaskshaped body (arrow) with adistinctive limiting membrane andcomplex internal substructures isalso apparent. The nucleus showsdiffusely dense chromatin, amarkedly irregular contour, and aprominent perinuclear electron-lucent halo. (Uranyl acetate-leadcitrate, x53 300).

of these forms elaborated distinctive spore-likecocci. The organisms were morphologically indis-tinguishable from the MLO that produced chronicorbital inflammatory disease in mice.' they producednuclear and cytoplasmic alterations similar to thosecaused by the other MLO.'-5 They parasitised asimilar wide range of leucocyte cell lines.'' Webelieve that these bacteria were MLO and caused theCOID in these patients.

Study of polymorphonuclear leucocytes facilitatesMLO detection. As in other chronic inflammatorydiseases,'5 polymorphonuclear leucocytes are presentin MLO diseases. Prominent cytoplasmic lysis

occurs in these leucocytes as a result of MLO parasit-isation.i The smallest MLO particles, namely, the0-005-O01 ism diameter filaments, are readilydetected in the lysed cytoplasm.3" Furthermore, cellwall material forming distinctive spore-like cocci isoften deposited in these cells.' Ah The lipid richpolymorphonuclear leucocyte cytoplasm' mayenhance MLO growth and development.'5

Chronicity was a noteworthy clinical feature of theMLO orbital disease in all three patients. Progressionto exophthalmos occurred in two of them. All hadchronic inflammation with various features includingvasculitis, tissue lysis, lymphoid infiltrates, and

868



j.com/

Br J O


ovember 1989. D

ownloaded from

http://bjo.bmj.com/

869Chronic orbital inflammatory disease: parasitisation oforbital leucocytes by mollicute-like organisms

Fig. 5 Several spore-like cocci areapparent. Undulatingpleomorphictubulospherical bodies (arrows),some with internal structures, arealso detectable. The cytoplasmshows patchy lysis and a paucity ofnormal granules. The nucleusdisplays several electron lucentchromatic foci of 'moth eaten'appearance, a ragged irregularcontour, and a perinuclear electronlucent halo. (Uranyl acetate-leadcitrate, x20 900).

granuloma formation. In the mouse orbit MLOinoculation produced similar chronic inflammation.The mouse disease was progressive, leading toexophthalmos, and vasculitis of small vessels was theinitial lesion.8 The mouse MLO orbital inflammatoryprocess resulted in tissue lysis, lymphoid infiltrates,and granuloma formation.8 Disease chronicity andprogression in the mouse was attributed to MLOdisruption of the host inflammatory process.""MLO were found in all three patients studied. A

search for MLO as a cause of COID in other healthypatients needs to be conducted to expand theseobservations. Moreover, the results of MLO experi-mental mouse disease suggest that MLO may be a

cause of human idiopathic systemic inflammatorydiseases in addition to orbital disease." "' COID

frequently occurs in association with chronic bowel,thyroid, and joint diseases. ' COID patients with suchassociated systemic diseases need to be investigatedfor MLO. The search should be conducted on boththe orbital and systemic disease.MLO diseases respond to long-term administration

of appropriate antibiotics.2 28 Rifampicin was tried inthe treatment of MLO experimental mouse diseaseand it significantly decreased the morbidity andmortality in comparison with untreated infectedcontrols.' Rifampicin was subsequently used to treathuman MLO uveitis unresponsive to corticosteroidsin uncontrolled studies.' With prolonged rifampicinadministration the ocular inflammation subsided,allowing reduction or discontinuation of corti-costeroid.'8 Rifampicin is a broad spectrum intra-



j.com/

Br J O


ovember 1989. D

ownloaded from

http://bjo.bmj.com/

870

cellularly acting inhibitor of bacterial DNA-dependent RNA polymerase and is safe for prolongedadministration." It seems to be indicated in thetreatment of orbital disease where MLO are demon-strated. Empirical administration of rifampicin maybe considered in COID patients where MLO cannotbe excluded by transmission electron microscopy oftissue leucocytes and the disease is progressive.

The authors are grateful to the physicians and technical staff of theEdward S Harkness Eye Institute, Columbia-Preshyterian MedicalCenter, Ness York, NY, whose co-operation enabled them toconduct this investigation.

References

I Jakohiec FA, Font RL. Noninfectious orbital inflammation. In:Spencer WH, Font RL, Green WR, Howes EL, Jakobiec FA,Zimmerman LE, eds. Ophthalmic pathology. 3rd ed. Phila-delphia: Saunders, 1986: 3: 2765-812.

2 Johnson LA, Wirostko E. Chronic idiopathic vitritis:ultrastructural properties of bacteria-like bodies withinvitreous leukocyte phagolysosomes. Am J Clin Pathol 1986; 86:19-24.

3 Johnson L, Wirostko E, Wirostko B. Chronic idiopathic vitritis:cytopathogenicity of unusual bacteria for vitreous polymorpho-nuclear leukocytes. J Submicro.s Cytol 1987: 19: 161-6.

4 Wirostko E, Johnson L, Wirostko W. Chronic leucocytoclasticbacterial Xitritis: a lymphocyte transmission electron microscopicstudy. J Suhnicrosc Cytol 1987; 19: 651-6.

S Wirostko E, Johnson L, Wirostko W. Chronic intracellularleucocytoclastic bacterial sitritis: a transmission electron micro-scopic study of the monocytes. J Submicrosc Cytol Pathol 1988;20: 463-7(0.

6 Johnson L, Wirostko E, Wirostko W. Crohn's disease uveitis:parasitization of vitreous leucocytes by mollicute-like organisms.Am J Clin Pathol 1989; 93: 259-64.

7 Wirostko E, Johnson LA, Wirostko BM. Transmission ofchronic idiopathic vitritis in mice by inoculation of humanvitreous containing leucocyte phagolysosomal bacteria-likebodies. Lancet 1986; ii: 481-3.

8 Wirostko E, Johnson L, Wirostko W. Mouse exophthalmicchronic orbital inflammatory disease. Virchows Arch (A) 1988;413: 349-55.

9 Johnson L, Wirostko E, Wirostko W. Mouse lethal cardio-vascular disease: induction by human leucocyte intracellularmollicutes. Br J Lrp Pathol 1988; 69: 265-79.

10 Wirostko E, Johnson LA, Wirostko WJ. Mouse interstitiallung disease and pleuritis induction by human mollicute-likeorganisms. Br J Exp Pathol 1988; 69: 891-902.

11 Freundt EA, Edward DG. Classification and taxonomy. In:Barile MF, Razin S, eds. The tnycoplasmas. New York:Academic Press, 1979 1: 1-41.


12 Starr MP. Foreward. In: Whitcomb RF, Tully JG, eds. Themycoplasmas. New York: Academic Press, 1979: 3: xi-xis.

13 Saglio PMH, Whitcomb RF. Diversity of wall-less prokaryotes inplant vascular tissue, fungi, and invertebrate animals. In:Whitcomb RF, Tully JG, eds. The Mycoplasmas New York:Academic Press, 1979: 3: 1-36.

14 Boatman ES. Morphology and ultrastructure of the Myco-plasmatales. In: Barile MF, Razin S, eds. The mycoplsmas. NewYork: Academic Press, 1979: 1: 63-102.

15 Rodwell AW, Mitchell A. Nutrition, growth, and reproduction.In: Barile M, Razin S, eds. The mycoplasmas. New York:Academic Press, 1979: 1: 103-40.

16 Smith PF. The composition of membrane lipids and lipopoly-saccharides. In: Barile M, Razin S. eds. The mycoplasmas. NewYork: Academic Press, 1979: 1: 232-57.

17 Barile MF. Mycoplasma-tissue cell interactions. In: Barile M,Razin S, eds. The mycoplasmas. New York: Academic Press,1979: 2:425-74.

18 Clyde WA. Mycoplasma pneumoniae infections of man. In:Tully J, Whitcomb R, eds. The mycoplasmas. New York:Academic Press, 1979: 2: 275-306.

19 Cassell GH, Hill A. Murine and other small-animal myco-plasmas. In: Tully J, Whitcomb R, eds. The mycoplasmas. NewYork: Academic Press, 1979: 2: 235-73.

20 Fernald GW. Humoral and cellular immune responses to myco-plasmas. In: Tully M, Whitcomb R, eds. The mycoplasmas. NewYork: Academic Press, 1979: 2: 399-423.

21 Horne RW. Comparison between the structure of animal andplant mycoplasmas: extracellular and intracellular morphology.In: Pathogeic mycoplasmas. Amsterdam: Associated ScientificPublishers, 1972: 39-66.

22 Bove JM. Mycoplasma infections of plants. lsr J Med Sci 1981:17: 572-85.

23 Ulanova EF. Changes in the nuclei and chloroplasts in cells of bigbud-infected tomatoes. Biol Bull Acad Sci USSR 1978: 5: 232-5.

24 Arora YK, Sinha RC. Enzymatic activities in cell fractions ofmycoplasmalike organisms purified from aster yellows-infectedplants. J Bacteriol 1985: 164: 811-5.

25 Marchesi VT. Chronic inflammation. In: Kissane JM, ed.Anderson's pathology. 8th ed. St Louis: Mosby, 1985: 1: 43-7.

26 Marmont AM, Damasio E, Zucker-Franklin D. Neutrophilmetabolism. In: Zucker-Franklin D, Greaves MF, Grossi CE,Marmont AM, eds. Atlas ofblood cells -function and pathology.2nd ed. Philadelphia: Lea and Fehiger, 1988: 1: 188-9(.

27 Sinha R. Chemotherapy of mycoplasmal plant diseases. In:Whitcomb R, Tully J, eds. The mscoplasmnas. New York:Academic Press, 1979: 3: 31(-35.

28 Wirostko E, Johnson L. The induction of mouse useitis byhuman idiopathic uveitis aqueous humor and treatment bycertain anti-tuberculous drugs. Trans NY Acad Sci ser 2, 1974:36: 693.

29 Mandell G, Sande M. Rifampin. In: Gilman A, Goodman L,RalI T, Murad F, eds. The pharmacological basis of therapeutics.7th ed. New York: MacMillan, 1985: 12(12-5.

Accepted for publication 25 May 1989.



j.com/

Br J O


ovember 1989. D

ownloaded from

http://bjo.bmj.com/

Documents

Chronic parasitisation mollicute-like · Chronic orbital inflammatory disease: parasitisation oforbitalleucocytesbymollicute-likeorganisms EMIL WIROSTKO,' LEWIS JOHNSON,2 AND BARBARAWIROSTKO1