RESULTS
Section 1
All samples were found to be positive for the anti-Ro antibody with higher than 240 IU/ml, except Sample ID # 13 and 32, which showed lower concentrations (Figure 1.). These samples also exhibited occurrence of Smd and Dn respectively, albeit at low concentrations. Sample Id# 12 and 20 also showed presence of anti-La antibody.
The autosera of Patient Id # 32 suffering from SS displays a faint 52kd IgM band, which is representative of 52kd anti-Ro antibody and is fainter in ethanol treatment sample. Patient ID # 42 expressing a higher than 240 IU/L of anti-Ro antibody in the clinical analysis, is a case of suspected SLE patient and shows a considerable presence of IgG’s of size 60, 80 and 100 kd and a faint band at 52 kd IgM band. Patient # 20 exhibits a robust expression of the 48 kd IgG for La and as is expected from the clinical report the concentration of La antibody is high in this patient suspected of Sjorgens syndrome. # 13 is a suspected SLE patient and displays several IgM bands at 48kd, 60 kd and 110 kd. # 12 is the autosera of patient with SLE, myositis, mouth ulcers and alopecia and as indicative from the clinical result, presents a distinctive expression of the 48 kd anti-La IgG antibody. # 41, which is a patient reporting fatigue and malaise displays absence of autoantibodies, whereas #48, undergoing radiotherapy for breast cancer and rash, indicates a faintly stained 60 kd IgM band, which may correspond to Ro 60 as anticipated from the clinical data. The clinical results from autosera of patient # 17 suggested presence of Ro 52, which can be reflected by presence of a 52 kd IgM band although of a lower intensity in the western blotting (Figure 2).
Figure 1. Samples are autosera obtained from humans suffering with autoimmune disorders like Sjorgen’s syndrome, SLE, patients suspected of SLE or SS, cutaneous Lupus monitoring test, patient with fatigue and malaise and patient undergoing radiotherapy everyday with rash. All samples were positive for the anti-Ro antibodies. Sample Id# 12 and 20 also demonstrated occurrence of anti-La antibody. Sample 13 and 32 also exhibited occurrence of other antibodies such as Smd and Dn respectively, albeit at low concentrations.
Figure 2: Western Blotting analysis of MCF-7 protein extracts with and without ethanol treatment. Key: SS- Sjorgens syndrome, SLE- systemic lupus erythematosus. Blue boxes indicate a difference in the intensities of ethanol treated and untreated control samples. Sample ID # 32 is a patient suffering from SS and displays a 52kd IgM band of lower intensity. Sample ID # 42 is a suspected SLE patient and show a considerable presence of IgG’s of size 60, 80 and 100 kd and a weaker 52 kd IgM band. # 20 exhibits a robust expression of the 48 kd IgG for La and as is anticipated from the clinical report, the concentration of La antibody is high in this patient suspected of SS. Sample ID # 13 is a suspected SLE patient and displays several IgM bands at 48kd, 60 kd and 110 kd. # 12 is the autosera of patient with SLE, myositis, mouth ulcers and alopecia and as indicative from the clinical result, presents a distinctive expression of the 48 kd anti-La IgG antibody. # 41, which is a patient reporting fatigue and malaise displays absence of autoantibodies, whereas #48, undergoing radiotherapy for breast cancer and rash, indicates a lower intensity 60 kd IgM band, which may correspond to Ro 60, as expected from the clinical data.
Section 2
Figure 3. represents a breast cancer patient (# 48), undergoing radiotherapy treatment and reports a rash. Biochemical test reveal presence of anti-Ro antibody with concentration > 240 IU/L. As anticipated from the clinical report, western blotting reveals presence of a 60 kd IgM band, albeit faintly stained. The band is slightly stronger under ethanol treatment, although needs further corroboration with more concentration of sample.
Effect of Ethanol
Ethanol treatment shows elevated expression of autoantibodies for certain bands of patient samples. For example, for patient # 13, the band intensity for ethanol treated samples are apparently higher than untreated samples, for IgM bands corresponding to 110 kd and 60 kd. Also, for patient ID 42, there seemed to be an overexpression of the ethanol treated protein corresponding to 80kd. For most of the other samples and protein bands, there is no apparent difference in the strength of fluorescence between the ethanol treated samples and control samples.
Figure 3. Representation of the western blotting using auto-sera from patient # 48, who is a 72-year-old female patient undergoing radiotherapy for breast cancer and developed a rash. The clinical report indicates presence of anti-Ro IgM autoantibody, although the blot reveals a low intensity of 60 kd bands. The band under ethanol treatment is stronger in intensity as compared to the control sample.
DISCUSSION
Section 1
In this study, western blotting confirmed the presence of 52kd IgM protein Ro 52, which was expected from the clinical results. Patient # 20 presented a distinctive expression of the 48 kd IgG for La and as is anticipated from the clinical report the levels of La antibody are apparently high in this patient suspected of Sjorgens Syndrome. As observed in Figure 2, patient # 12 represents autosera of patient with SLE, myositis, mouth ulcers and alopecia and as is indicative from the clinical result, presents a distinctive expression of the 48 kd anti-La IgG antibody. This finding is in line with the presence of anti-52 antibodies demonstrated to be typically associated with myositis . Anti-La has been frequently observed in polymyositis and SS overlap syndrome, occurring in about 1-14 % of the patients. Anti-La also called anti-SSB is present in 15 % of patients suffering from SLE and other disorders of connective tissue like SS, also linked to neonatal lupus .
In this study, patient Id # 48 is a breast cancer patient undergoing radiotherapy and exhibits occurrence of rash. A rash is a typical symptom occurring in autoimmune diseases like SLE . Earlier association of autoantibodies with malignancies and particularly breast cancer, makes this autosera sample interesting. Occurrence of a IgM band, albeit of lower intensity, renders this sample interesting and worth exploring further for more analysis.
Section 2
Ethanol treatment caused overexpression of some protein bands such as the IgM bands in sample #13 and 42, which may be attributable to experimental errors in protein loading. For most other samples and proteins there seemed to be no significant difference in the band intensities between control and ethanol treated samples. The observed difference may be corroborated with more number of repetitions of the experiment in replicates.
The findings of this study confirm the presence of IgG and IgM autoantibodies in the analyzed samples, corresponding to Ro 60, Ro 52 and Ro 48. Ethanol treatment demonstrated elevated expression for some proteins, which may be experimental artifacts and need further corroboration.
Section 3
Elevated generation of IgM has been found to occur in patients suffering from SS . About 60-80 % of the patients diagnosed with SS exhibit as Ro-positive . Ro52 may directly trigger activation of autoimmune T cells and B cells in SS. There is evidence of about two-fold rise in the Ro52 expression in patients of SS as compared to healthy control subjects .
Autoantibodies targeted to a gamut of cellular components have been found in autoimmune diseases like Sjorgen’s syndrome and SLE. Anti-SS-A (Ro 60) and anti-SS-B (La 48) are particularly detected in SLE and Sjogren’s syndrome . Anti-Ro antibodies have been demonstrated to be expressed in SLE, diseases with an overlap of SS and SLE, whereas anti-La is more specifically associated with SS .
Autoantibodies specific to small nuclear ribonucleoprotein complexes have been demonstrated in patients suffering from SLE and other associated disorders. These antibodies, typically target spliceosomal proteins and are a characteristic feature of SLE. One such autoantibody, Smd was detected in the autos era of patient # 13, suspected of SLE. Anti-Sm antibodies are highly specific to SLE and detected in 5-30 % patients .
While occurrence of antinuclear antibodies has been demonstrated in many malignancies, some studies have reported controversial findings. A case control study suggested the prevalence of ANA’s in malignancies, including breast cancer, even absence of autoimmune diseases.. Researchers in the past have reported presence on autoantibodies in the sera of patients with carcinoma of the breast. Higher ANA’s in breast carcinoma . A recent report presents compelling evidence for occurrence of autoantibodies in sera of breast cancer suffering women or benign breast disease and showed that these antibodies present novel immunologic characteristics mirroring the rheumatic autoimmune diseases . These studies open up important avenues for establishment of panels of breast-cancer associated autoantibodies.
While there is evidence indicating the presence of autoantibodies in autosera of patients suffering from breast cancer and these antibodies show immunologic properties resembling rheumatic diseases, no studies have explored specificity of autoantibodies in autosera of systemic autoimmune diseases, towards proteins in breast cancer MCF7 cell line. This study is an important step in this direction and shows occurrence of many IgG and IgM autoantibodies recognizing breast cancer proteins. Ethanol treatment showed a difference in the expression of some proteins of a few autosera samples, but may not be concluded as significant. The data needs replication with more number of samples and quantification to confirm this difference.
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