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ORIGINAL ARTICLE
Year : 1997  |  Volume : 45  |  Issue : 2  |  Page : 93-97

Evaluation of lymphocyte proliferation assay to purified protein derivative, enzyme linked immunosorbant assay, and tuberculin hypersensitivity in Eales' disease


Medical and Vision Research Foundation, Chennai, India

Correspondence Address:
J Biswas
Medical and Vision Research Foundation, Chennai
India
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Source of Support: None, Conflict of Interest: None


PMID: 9475026

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  Abstract 

The purpose of this study was to evaluate the immunological responses against mycobacterial antigens in Eales' disease. Fifty six patients with Eales' disease and fifty age-and-sex-matched healthy volunteers with normal fundus findings taken as controls, were subjected to Mantoux test, using 2 TU/0.1 ml of purified protein derivative (PPD), lymphocyte proliferation assay to PPD, and ELISA to detect IgM and IgG antibodies against mycobacterial A-60 antigen. The results of Mantoux test and lymphocytes proliferation assay did not differ significantly in the two groups suggesting a similar cellular immune response. The number of individuals with recent exposure/reexposure to tuberculosis (IgM+) was significantly higher among patients. However the number of people with past exposure (IgM-IgG+) was significantly higher among controls. Our study indicates that there are no statistically significant differences in the humoral and cellular immune responses to mycobacterial antigens between the patients with Eales' disease and controls, except for a significantly higher IgM positivity among the patients.

Keywords: Eales′ disease, ELISA, Lymphocyte Proliferation Assay, Tuberculosis, Mantoux test


How to cite this article:
Biswas J, Narain S, Roy S, Madhavan H N. Evaluation of lymphocyte proliferation assay to purified protein derivative, enzyme linked immunosorbant assay, and tuberculin hypersensitivity in Eales' disease. Indian J Ophthalmol 1997;45:93-7

How to cite this URL:
Biswas J, Narain S, Roy S, Madhavan H N. Evaluation of lymphocyte proliferation assay to purified protein derivative, enzyme linked immunosorbant assay, and tuberculin hypersensitivity in Eales' disease. Indian J Ophthalmol [serial online] 1997 [cited 2021 Mar 6];45:93-7. Available from: https://www.ijo.in/text.asp?1997/45/2/93/15010



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Eales' disease is recognized as a primary retinal vasculitis of unknown aetiology occurring in otherwise healthy adults, mostly men, between the age of 15 and 40. The disease is mostly seen in India, Pakistan and Afghanistan[1]. In India the incidence of Eales' disease has been reported to be 1.35% among the ophthalmic patients at a referral eye center[2]. Several studies have been undertaken to explore the role of infectious, endocrinal, biochemical and immunological factors in an attempt to understand the aetiopathogenesis of the disease. Among the several infectious aetiologies, tuberculosis has been implicated by Axenfold and Stock[3] and supported by several other workers. The assumption of tubercular aetiology was based on the observation of active or healed tuberculosis in patients with Eales' disease. However the incidence of Eales' disease in Indian patients with tuberculosis has been found to be rather low. In one series, only 1.3% of patients had Eales' disease[4]. In our earlier studies none of the 100 cases with active pulmonary or extrapulmonary tuberculosis, or the 110 cases with healed tuberculosis, had Eales' disease[5],[6]. The high incidence of positive Mantoux test (upto 90% in some series[7][8][9]) had led some to believe in the possible role of an allergic reaction to tuberculoprotein, but a similar high incidence in the normal population raised serious doubts regarding the validity of such an interpretation[9]. Based on a study of immunological parameters including the humoral responses to BCG antigen, a possible mediation through type III (immune complex mediated), and/or type IV (delayed) hypersensitivity reaction, has also been suggested[10]. However there is no report or evidence supporting the occurrence of a systemic vasculitis in Eales' disease as seen in other vasculitic entities due to type III hypersensitivity state. Thus selective sensitization of retinal tissue by tuberculoprotein has been suggested as a mechanism for tubercular aetiology[12].

We reevaluated the role of tuberculosis in Eales' disease by comparing the humoral immune response to mycobacterial A-60 antigen, lymphocyte proliferation assay and cutaneous response to purified protein derivative (PPD) in 56 patients with Eales' disease and 50 healthy, age-and-sex-matched controls.


  Materials and methods Top



  Patients and Controls Subjects: Top


All unrelated new patients of either sex, between 15 and 40 years attending the out-patient department of a major referral eye center in Madras, who presented with at least one of the three inclusion criteria: (1) Peripheral retinal periphlebitis in any eye; (2) Neovascular or fibrovascular proliferation at the level of retina, or elevated into the vitreous cavity with or without tractional retinal detachment with periphlebitis in the same and /or other eye; and (3) Vitreous haemorrhage with periphlebitis in the same or other eye, scrutinized to rule out conditions mimicking Eales' disease were recruited. For this purpose a battery of laboratory tests comprising of a haemogram, electrophoresis for sickle cell trait, rheumatoid factor, antinuclear antibodies, LE cell phenomenon, venereal disease research laboratory test (VDRL), Treponema pallidum haem-agglutination test (TPHA), serum angiotension converting enzyme, Mantoux test, chest X-ray and a postprandial blood sugar were performed. Patients with periphlebitis due to secondary causes such as syphilis, sarcoidosis, collagen disorders or sickle cell retinopathy were excluded. Patients with active or healed choroiditis, diabetes mellitus, central or branch retinal vein occlusion without periphlebitis and those who had undergone a photocoagulation, cryotherapy or a vitreous surgery prior to presentation, were excluded. Fifty six consecutive patients with Eales' disease and 50 age-and-sex-matched controls were finally enrolled. The controls also underwent a complete ocular examination and investigations to rule out intraocular inflammation, perivasculitis or lesions mimicking Eales' disease.


  ELISA Technique Top


ELISA was done by a standard protocol for detection of IgM and IgG antibodies against mycobacterial A-60 antigen using a commercially available ELISA kit (Anda Biologicals, France).


  Mantoux Test Top


Mantoux test (MT) was done by injecting 2 TU/0.1 ml of tuberculin PPD supplied by BCG laboratory, Madras, India. The results were read after 48 hours. An induration ≥ 10 mm was considered as positive.


  Lymphocyte Proliferation Assay Top


This test was carried out as described by Maluish and Strong[12]. Brief description of the test is as given below: Peripheral blood (10 ml) was collected by venipuncture into a sterile tube containing 500 IU of preservative free sodium heparin. The whole blood was diluted with equal amount of sterile PBS at pH 7.3. The seperation of mononuclear cells was done by density grandient centrifugation of whole blood. For that, the diluted whole blood was carefully overlaid onto 3 ml of lymphoflot (density gradient solution) in a sterile glass centrifuging tube and centrifuged for 30 minutes at 1500 rpm. Lymphocytes seperated as a white band between the plasma and lymphoflot were carefully pipetted out into a sterile container and counted in the Neubaur counting chamber. Cells were diluted with Eagle's MEM supplemented with 5% foetal calf serum such that one microlitre contained 2000 cells. All cultures were done in duplicates. 96 well tissue culture plates were used. Lymphocyte cell suspension and Eagle's MEM in 100 (μl volumes and phytohemagglutinin (PHA) and PPD in 10 μl volumes were added as per protocol given below in 16 wells:

Plates were incubated at 37C with 5% CO2. On the third day 10 μl of tritiated thymidine (BRIT, Bombay) was added to the control set that is 12 to 14 hours before harvesting. On fourth day, the counting of the cells were done in a scintillation counter (Beckman LS 6000). For that, the medium was pipetted out from the wells, the cells were generally washed with sterile normal saline thrice, one drop (approximately 50 μl of Trypsin EDTA added into each well. After 2 minutes the cells were transferred into 4 ml of scintillation fluid for each well and were shaken and loaded in the scintillation counter and the reading in DPM was made. On the sixth day tritiated thymidine was added to the test wells and readings were taken on the seventh day same as above. PHA which nonspecifically induces proliferation was included in the tests as controls for the verification of response of T lymphocytes. Stimulation Index was calculated as DPM Value of (cells + PPD) divided by the DPM value of cell control.


  Results Top


The results of ELISA for IgM and IgG antibodies against the mycobacterial A-60 antigen are presented in [Table - 1]. The number of individuals with positive IgM antibodies was significantly higher (29.2%) among the patients than among the controls (12.5%). There was no statistically significant difference in the positivity for the IgG antibodies in the two populations.

ELISA results were further analysed by dividing them into four classes depending upon the detection of one or both of IgM and IgG antibodies. The number of individuals who showed negative result for both the antibodies was 16 (33.3%) and 10 (20.8%) among the controls. Five patients (10.4%) and three controls (6.2%) had only IgM antibodies. Nine patients (18.8%) and three controls (6.2%) were positive for both the antibodies. The difference in the numbers of people belonging to these three classes was not statistically significant. However the number of individuals belonging to IgM-, IgG+ class was significantly higher (32,66.7%) among the controls, than among the patients (18,37.5%) [Table - 2]. Overall seropositivity rates, based on detection of at least one of the two antibodies were 66.7% (32 patients) and 79.2% (38 controls). The difference was insignificant.

The results of the Mantoux test as shown in [Table - 3] were statistically not significant in the two groups with 89.3% patients and 75% controls positive. Similarly the numbers of individuals in the two classes with positive lymphocyte proliferation assay [Table - 4] i.e. with a stimulation index of one or more, were similar (71.4% of patients and 74% of controls). The average stimulation index (SI) for the patients was 1.30 0.78 standard deviation (SD) and for the controls 1.32 0.60 (SD). Using t-test, and difference of the SI between the two groups was not found to be significant (p = 0.91).

The correlation between Mantoux test and lymphocyte proliferation assay was studied as is shown in [Table - 5]. The various permutations had similar frequencies in the two populations. The number of individuals with dissociation between these two was the same (14 out of 48) in the two populations. Among the patients 11 individuals were positive for Mantoux test but negative for lymphocyte proliferation assay. The corresponding number among the controls was 7. Three patients and 7 controls were positive only for lymphocyte proliferation assay and not Mantoux test. These differences were not significant. Finally we studied the correlation between the results of ELISA, Mantoux test and lymphocyte proliferation assay, as summarised in [Table - 6]. There was no statistically significant difference in this analysis either.

Since not all patients and controls could be tested for all the immunological tests for various reasons, we analysed the maximum possible numbers of individuals for each set of tests.


  Discussion Top


In the present study, humoral response to tubercular antigen was determined by the ELISA test, and cellular immune response was determined by the Mantoux test and lymphocytes proliferation assay. The ELISA test was done by using the A60 antigen. The A60 is a complex of antigens containing both free and bound lipids, proteins and polysaccharides[13]. It is the main thermostable component of old tuberculin and purified protein derivative of tuberculin[14], and has a prominent immunologic role[15]. Thus its use provides an opportunity to study the humoral immune response to the same antigens or their epitopes, which as constituents of PPD are used to study the cellular immune response. However the sensitivity of specificity of this ELISA kit has not been proven to be high enough to use as a diagnostic test for systemic tuberculosis infection and should be used in conjuction with other diagnostic means, that together will allow the determination of a diagnosis[16].

Gevaudan and coworkers[17] in a study from France found, out of 344 cases of primary tuberculosis 88% were positive for anti A-60 IgG and 75% for the corresponding IgM. Among 97 cases of primary extrapulmonary tuberculosis, 94% were IgG positive and 33% were IgM positive. The difference between active and inactive post-primary (chronic) tuberculosis was striking. About 100% of both pulmonary and extrapulmonary cases (367 altogether) had high titers of anti A-60 IgG but IgM positivity was observed in only 15% of the cases, whereas in inactive and quiscent non cavitary tuberculosis (442 cases), 57% of the patients were weakly positive for anti A-60 IgG and none were positive for IgM. Among patients suffering from nontuberculosis disease 25.7% were positive for anti A-60 IgG and 4.6% were IgM positive. In our series both IgM, and IgG negative group indicates no antibody response against A-60 antigen in 33.3% of patients and 20.8% of the controls. This group indicates that humoral antibody response is absent in significant percent of Eales' disease patients and most likely do not have any active tuberculosis infection unless there is a small possibility of anergy exists. Other combinations of ELISA result against A-60 antigen indicates that similar response to controls exists. Similar response in cases and controls indicate that humoral response to mycobacterial antigen may not be responsible for pathogenesis of Eales' disease. We had only two statistically significant findings in the comparative analysis of the humoral immune responses of the two populations. The first of these was a significantly higher number of individuals positive for IgM antibodies among patients, suggesting a recent exposure/reexposure to tuberculosis, and a consequent primary response. The second observation was that a higher fraction of the controls were negative for IgM antibodies but positive for IgG antibodies. This subclass represented those people who had been exposed to tuberculosis in the past and had memory cells to produce IgG antibodies.

The observation that a significantly higher number of controls belonged to IgM-, IgG+ subgroup, simply underlines the greater number of controls than patients who were possibly exposed to tuberculosis in the past and were now generating IgG antibodies either in low baseline titres due to a secondary immune response, or in high titers due to a subsequent rechallenge. The actual number of people ever exposed to tuberculosis, that is those positive for either IgM or IgG antibodies, did not differ statistically. These observations further undermine the presumed aetiological role of tuberculosis. A similar analysis of cellular immune response to tubercular antigens also did not support the hypothesis of tubercular aetiology in Eales' disease, as 45 out of 56 cases and 43 out of 50 controls were positive for at least one of the two tests: the Mantoux test and the lymphocyte proliferation assay.

The delayed hypersensitivity reaction to the tuberculoprotein as incriminated in the past is unlikely to be an aetiological factor for Eales' disease as there was no statistically significant difference between the cellular immune responses between patients and controls. This is consistent with the one of the author's earlier observations where no case of Eales' disease was seen in an extensive clinical and laboratory evaluation of 1005 patients with active pulmonary or extrapulmonary tuberculosis[5]. As stated earlier, only 1.3% of patients with active or healed tuberculosis had Eales' disease in another series[4] which is a rather weak association. Thus tuberculosis may either be an incidental association or just one of the several 'environmental factors'. Had it been an important causative factor, it would be difficult to explain the incidence of Eales' disease among patients with a negative Lymphocyte proliferation assay, Mantoux test and negative humoral antibody response to Mycobacterial antigens. Earlier studies based on the light microscopy and immunohistochemistry of epiretinal membrane (ERM) and subretinal membrane (SRM) of patients with Eales' disease have demonstrated predominant T-cell involvement in the lymphocytic infiltration[18],[19], suggestive of a role of cell mediated immune dysfunction. However our study did not show any difference in the cellular immune response to tubercular antigens; the only observed differences were in the humoral immune responses which neither parallel the results of the immunohistochemical studies nor support a tubercular aetiology. Furthermore there is no support for the tubercular association with Eales' disease even from animal models[20].

In summary, at this stage there is no evidence, except a statistically significant difference in the IgM positivity rates, to believe that tuberculosis is an aetiological factor for Eales' disease. Any association with tuberculosis may merely be incidental as may have been several other associations with non tubercular entities studied in the past.

 
  References Top

1.
Gieser SC, Murphy RP: Eales' disease. In Ryan S (ed): Retinal disease, St. Louis, C.V. Mosby, 86:53-539, 1989.  Back to cited text no. 1
    
2.
Badrinath SS, Honnatti MR: Vitrectomy in Eales' disease: Acta Int Cong Ophthalmol., 24:536, 1982.  Back to cited text no. 2
    
3.
Axenfold and Stock.XI Int. Cong, ophthalmol., Naples, 367, 1909.  Back to cited text no. 3
    
4.
Awasthi P, Mehtotra ML, Srivastava SN: Ocular conditions in pulmonary tuberculosis patients in India. Acta XX Concilium Ophthalmologicum. 1025, 1966.  Back to cited text no. 4
    
5.
Biswas J: Ocular morbidity in patients with active systemic tuberculosis. Nethralaya Insight. 4:3-6, 1988.  Back to cited text no. 5
    
6.
iswas J: Ocular morbidity in patients with healed systemic tuberculosis. Nethralaya Insight. 4:7-8, 1988.  Back to cited text no. 6
    
7.
Elliot AJ: Recurrent intraocular haemorrhage in young adults (Eales' disease). A report of 31 cases. Trans Am Ophthalmol Soc. 52:811, 1954.  Back to cited text no. 7
    
8.
Donders PC: Eales' disease. Doc Ophthalmol. 12:1-105, 1958.  Back to cited text no. 8
    
9.
Raj N, Krishnamurthy MS, Anantharaman DS: Prevalence of non-specific sensitivity in some parts of India. Ind J Med Res 63:1098, 1975.  Back to cited text no. 9
    
10.
Muthukkaruppan VR, Rengarajan K, Chakkalath HR, et al: Immunological status of patients of Eales' disease. Ind J Med Res. 90:351-359, 1989.  Back to cited text no. 10
    
11.
Ashton N: Pathogenesis and aetiology of Eales' disease. Acta XIX Concilium Ophthalmologicum 2:1962.  Back to cited text no. 11
    
12.
Maluish AE, Strong DM: Lymphocyte proliferation in Manual of clinical laboratory immunology, (ed.) Rose NR, Friedman H, Fabey JL, 3rd Edition, American Society for Microbiology, Washington DC. 1986.  Back to cited text no. 12
    
13.
Fabre I, L'Homme O, Brunteau M, et al. Chemical composition of antigen A60 from Mycobacterium bovis BCG.Clin Exp Immunol 66:262-72, 1986  Back to cited text no. 13
    
14.
Harboe M. Antigens of PPD, old tuberculin and autoclaved M. bovis BCG studied by crossed immunoelectrophoresis. Am Rev Respir Dis 124:80-87, 1981  Back to cited text no. 14
    
15.
Harboe M, Closs O, Bjorvatn B, Bjung G. Antibodies against BCG antigen A60 in mycobacterial infection. Br Med. J, 2:430-433, 1977  Back to cited text no. 15
    
16.
Cocito CG: Properties of the mycobacterial antigen complex A-60 and its applications to the diagnosis and prognosis of tuberculosis. Chest, 100:1687-1693, 1991.  Back to cited text no. 16
    
17.
Gevaudan MJ, Bollet C, Charpin D, et al. Serological response to tuberculosis patients to antigen 60 of BCG. Eur J Epidemiol. 8:666-676, 1992.  Back to cited text no. 17
    
18.
Biswas J, Rao NA: Epiretinal membrane in Eales' disease and other vascular retinopathies. Invest Ophthalmol Vis Sci. 31 (Suppl): 369, 1990.  Back to cited text no. 18
    
19.
Badrinath SS, Biswas J,Sharma T, et al: Immunophenotyping of inflammatory infiltrates in epiretinal (ERM) and subretinal membrane (SRM) in Eales' disease. Invest Ophthalmol Vis Sci.33 (Suppl):857, 1992.  Back to cited text no. 19
    
20.
Finoff WC: Ocular tuberculosis, Experimental and clinical. Arch Ophthalmol. 53:130-136, 1924.  Back to cited text no. 20
    



 
 
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  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7]



 

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