Ligneous conjunctivitis: a clinicopathological, immunohistochemical, and genetic study including the treatment of two sisters with multiorgan involvement
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- Rodríguez-Ares, M.T., Abdulkader, I., Blanco, A. et al. Virchows Arch (2007) 451: 815. doi:10.1007/s00428-007-0481-9
Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, β2-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results.
KeywordsLigneous conjunctivitisPlasminogen geneNasal polypsGastric ulcerMutation analysis
The term ligneous conjunctivitis (LC) was coined in 1933 by Borel  to describe a rare form of chronic, bilateral, recurrent conjunctivitis characterized by the development of firm fibrin-rich, woody-like pseudomembranous lesions mainly on the upper tarsal conjunctivae, and less frequently in the lower eyelid and the bulbar conjunctive . LC was first described by Bouisson  as early as 1847, and the microscopic appearance of pseudomembranes presented as inflammatory fungus-shaped masses first reported in 1924 . Because similar lesions can occur on other mucous surfaces indicating a systemic disease, Mingers et al.  proposed the term pseudomembranous disease for this entity; the same group  also demonstrated severe inherited type I plasminogen (PLG) deficiency as the cause of this disease in 1994. Familial occurrence was also reported [4, 38], and distinct homozygous and compound-heterozygous mutations in the PLG gene have been demonstrated by Schuster et al. [35, 39] and other groups since 1997.
Extraocular lesions were reported occurring in the oral cavity (mainly gingiva) [14, 40], the upper gastrointestinal tract , the ear (including temporal bone) [7, 15], the nasopharynx, larynx, tracheobronchial tree, and lung [10, 27], the female genital tract (sometimes with infertility) [8, 17, 28], and the renal collecting system . A new syndrome or a coincidental association of non-Hodgkin’s lymphoma, auricular hypoplasia, and juvenile colloid milium with LC were recently noted . In very rare cases, congenital occlusive hydrocephalus has been associated with the more severe forms of the disease and may even precede LC [1, 10, 36]. More recently, the report of a single patient with extensive woody deposits in her eyes, hydrocephalus, gingival hyperplasia, otitis media, sinusitis, and genital tract involvement exemplified the various manifestations of the pseudomembranous disease characteristic of severe PLG deficiency .
In 1998, it was shown that repeated infusions of PLG could resolve ligneous pseudomembranes , and effective treatment with topical PLG was reported in 2002 . More recently, amniotic membrane transplantation has shown promising results in conjuntival reconstruction in difficult-to-manage cases of LC [2, 3].
In this paper, we report the clinicopathological, immunohistochemical, and genetic data of two sisters that presented with LC in adulthood, one of whom was treated with amniotic membrane transplantation.
Two sisters of 57 (Case 1) and 62 (Case 2) years of age were referred to the Clinical University Hospital with a diagnosis of bilateral epidemic conjunctivitis and unsatisfactory responses to different treatments. Consanguinity and moderate deafness were found in both patients. In Case 2, there had been a previous history of deafness at age 32, recurrent gastric peptic ulcer at age 57, and recurrent nasal polyps at age 60. No other additional relevant data were found in the clinical records of the two patients. The patients showed abundant mucopurulent secretions with wood-like pseudomembranes on the upper and lower tarsal conjunctiva, with Staphilococcus epidermidis in culture.
Materials and methods
Histological, histochemical, and immunohistochemical analyses
The surgical specimens were fixed in buffered formaldehyde and routinely embedded in paraffin. The 4-μm-thick sections were stained with hematoxylin and eosin and Congo red stain. Immunohistochemical studies were also performed on paraffin sections using a universal second antibody kit that utilized a peroxidase-conjugated labeled-dextran polymer (Dako EnVision Peroxidase/DAB; Dako, Glostrup, Denmark), to avoid misinterpreting endogenous biotin or biotin-like activity in tissues  as positive staining. The following antibodies were used: amyloid A component (clone mc1, dilution 1:50, Novocastra, Newcastle upon Tyne, UK), transthyretin (polyclonal, 1:1,000, antigen retrieval water bath, Dako, Glostrup, Denmark), β2-microglobulin (polyclonal, 1:2,000, Dako), fibrinogen (polyclonal, 1:200, microwave, Dako), albumin (polyclonal, 1:2,000, BioGenex, San Ramon, CA, USA), fibronectin (polyclonal, 1:10,000, water bath, Dako), collagen type IV (CIV, 1:10, microwave oven and EDTA), vimentin (V9, 1:5,000, microwave oven, BioGenex), cytokeratins (AE1–AE3, 1:20, water bath and proteinase K, Dako), immunoglobulin A (IgA; polyclonal, 1:10,000, microwave oven, Dako), IgD (polyclonal, 1:1,000, water bath, Dako), IgG (polyclonal, 1:10,000, microwave oven, Dako), IgM (R1/69, 1:500, microwave oven, Dako), light chains kappa (polyclonal, 1:50,000, water bath, Dako), light chains lambda (polyclonal, 1:50,000, water bath, Dako), CD3 (SP7, 1:50, water bath and EDTA, Master Diagnóstico, Granada, Spain), CD4 (4B12, 1:4, water bath and EDTA, Master Diagnóstico), CD8 (4B11, 1:20, water bath and EDTA, Novocastra), and CD20 (L26, 1:50, water bath, Dako). Negative control samples (the primary antibody was replaced by nonimmune mouse serum) and positive tissue samples were included in each slide run.
Informed consent was obtained for this study, which was conducted according to the Spanish law including adherence to the Helsinki Principles of 1975, as revised in 1983. For Case 1, genomic DNA was extracted from peripheral white blood cells by standard methods. For both cases (Case 1 and Case 2), to confirm our results, genomic DNA was extracted from paraffin-embedded tissue using QIAamp DNA Kit and QIAamp DNA Blood Mini Kit (QIAGEN: www.qiagen.com), following the manufacturer’s instructions. Thereafter, all coding regions and exon/intron boundaries of the PLG gene were amplified by polymerase chain reaction (PCR). To prevent the coamplification of homologous genes, we used primers and strategy described in Siboni et al. . After the amplification, PCR products were purified. Both strands were subjected to cycle sequencing with the same PCR primers that covered the complete gene, both exons and introns, using the BigDye terminator kit and run in the 3730 xl DNA Analyser (Applied Biosystems, Foster City, CA, USA).
Pathological and immunohistochemical findings
Reevaluation of biopsies in Case 2 showed both exuberant granulation tissue and subepithelial fibrin deposits in gastric biopsies (Fig. 2d) and in metastatic vaginal lesions from the ovarian adenocarcinoma. The nasal polyps were also composed of subepithelial deposits of amorphous eosinophilic material instead of the loose mucoid stroma characteristic of nasal inflammatory polyps (Fig. 2c).
According to the excellent revision of Shuster and Seregard , the diagnosis of LC is based on the clinical picture (pseudomembranous lesions), the typical histological findings, and eventually on a positive family history. All of these criteria were present in the two patients here presented, with some unusual features that deserve consideration.
The prevalence of LC may be roughly estimated in the range of 1.6 per 1 million people , at least in Europe, but in countries where consanguinity is more frequent, the prevalence of LC may be higher . Although most cases of LC are sporadic, some familial cases have been reported with an autosomal-recessive pattern [4, 38]. In most cases, infants and children are affected, but late onset manifestation of LC may occur, as in our patients, ranging from birth to 85 years [18, 38]. Both eyes are affected in up to 51% of cases, and corneal involvement, a complication that may lead to blindness, occurs in 20–30% of cases . Systemic signs and symptoms may precede or concomitantly occur with the ocular disease.
Histopathological findings of the ocular and extraocular lesions in LC are identical. The excessive depositions of fibrinogen in LC should be differentiated from very rare cases of amyloidosis of the eye . While amyloid deposits are positive with the Congo red stain, fibrinogen is negative. It must be noted that, in some extraocular biopsies, the nature of the disease may be overlooked by the pathologist, particularly if there is no clinical suspicion of LC, as occurred in our Case 2. Immunohistochemical studies for fibrinogen can definitively confirm the nature of the eosinophilic material in LC as occurred in our biopsies.
PLG plays an important role in fibrinolysis and wound healing. In patients with LC, wound healing in mucous surfaces is impaired and seems arrested at the stage of granulation tissue [30, 38]. Histopathological and immunohistochemical findings indicate that fibrinogen is the major constituent of the membranes in LC. As was referred to in the literature, and as we confirmed in our patients, severe type I PLG deficiency (hypoplasminogenemia) is a major cause of LC [23, 24, 38]. The observation of cases of LC developed during treatment with tranexamic acid, an antifibrinolytic drug, also supports this idea .
Several mutations have been detected in the PLG gene of patients affected with LC [10, 33, 38, 43, 45]. PLG is the proenzyme of the serine protease plasmin and is a 791-amino-acid residue single-chain glycoprotein with a molecular weight of 93 kDa . Its gene spans approximately 52.5 kb on chromosome 6q26–27 and consists of 19 exons . The proteolytic activity of plasmin contributes to the digestion of the insoluble fibrin clot and thrombi by which normal recanalization and tissue repair can be accomplished . The mutation p.Thr600Ile of the PLG gene is very close to His-603, one of the three amino acid residues that constitute the serine protease triad. Moreover, p.Thr600Ile is next to p.Ala601Thr, a mutation present in 2–4% of Japanese subjects that predisposes them to thrombophilia and is known as Plasminogen Tochigi . But, as stated for ligneous conjunctivitis , the mutation found in our patients does not predispose them to thromboembolic episodes . In these patients, however, the risk of thrombotic occlusions of implanted catheters may be markedly increased [21, 38]. To the best of our knowledge, this is the first report of this mutation described in patients with LC.
LC has also been reported in different animal species ; mice in which both PLG genes have been knocked out (PLG-deficient mice, Plg−/− mice) will develop conjunctival lesions indistinguishable from human lesions . The clinical phenotype in Plg−/− was reported to differ from that in humans with LC, in that, in humans, gastric, colon, and rectal ulcers, rectal polyps, and liver involvement have not been reported so far. We believe, however, that our Case 2 patient is, as far as we know, the first description of recurrent gastric ulcer and LC.
Shuster and Seregard  and Tefs et al.  concluded that a potentially useful initial approach for LC is the topical application of a PLG concentrate, possibly in combination with a PLG activator (uPA or tPA), which may soften the pseudomembranes and facilitate removal. To prevent recurrence, manipulations of the conjunctiva should be kept to a minimum, and treatment with heparin and corticosteroids should be undertaken, sometimes combined with topical cyclosporine A [3, 38, 44]. Topical and subconjunctival fresh frozen plasma may also help in the prevention of membranes in susceptible PLG-deficient patients . Therapy with intravenous infusion of lys-plasminogen was favorable in one case , but impossible as a long-term solution due to the short half-life of the preparation, the risk of intravascular fibrinolysis activation, and the high costs . In rare cases, LC may resolve itself spontaneously . Amniotic membrane transplantation is effective in some corneal and conjunctival lesions because of production of antiangiogenic and antiinflammatory proteins; Barabino and Rolando [2, 3] has proposed this as a promising therapy for LC. Although other experimented researchers  have not found this procedure adequate, we have had no recurrences of pseudomembranes in our patient for 39 months after amniotic membrane transplantation nor did any appear in the patient reported by Barabino and Rolando  for at least 3 years. Oral contraceptive therapy deserves attention as an alternative therapy in selected cases of LC .
In summary, the present paper, describing two cases of LC shows that LC is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to PLG deficiency. Pseudomembranes are subepithelial deposits of amorphous eosinophilic material (fibrinogen) with a variable proportion of granulation tissue and inflammatory cells. The same subepithelial deposits occurred as nasal polyps and recurrent ulcers in gastric mucosa. A novel homozygous point mutation c.1856 C>T in exon 15 of the PLG gene was found in both patients, and amniotic membrane transplantation was done in one case with promising results.
We thank Ms. Elena Couso Folgueiro for her technical assistance.