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Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens.

Talwar H, Rosati R, Li J, Kissner D, Ghosh S, -Madrid FF, Samavati L - EBioMedicine (2015)

Bottom Line: Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients.Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls.These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Wayne State University School of Medicine and Detroit Medical Center, Detroit, MI 48201.

ABSTRACT

Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB). No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

No MeSH data available.


Related in: MedlinePlus

Venn diagram depicts differential phage clone significances among sarcoidosis, TB and healthy controls (q < 0.01). Venn diagram shows the overlap between 259 sarcoidosis clones and 238 TB clones as compared to healthy controls, as well as 380 TB clones versus sarcoidosis. Forty seven clones could differentiate both sarcoidosis and TB from healthy controls. Five clones could not discriminate between TB and sarcoidosis.
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f0020: Venn diagram depicts differential phage clone significances among sarcoidosis, TB and healthy controls (q < 0.01). Venn diagram shows the overlap between 259 sarcoidosis clones and 238 TB clones as compared to healthy controls, as well as 380 TB clones versus sarcoidosis. Forty seven clones could differentiate both sarcoidosis and TB from healthy controls. Five clones could not discriminate between TB and sarcoidosis.

Mentions: In view of the clinical and pathological similarities between MTB and sarcoidosis, a most useful clinical antigen(s) should discriminate between these two conditions. To this end, using the antigens identified by biopanning the CSL library, a microarray was constructed and then this construct was interrogated with sera from 17 culture positive MTB subjects. Using a moderate t-test and a q value of < 0.05 in this system, we identified 238 clones differentially expressed between TB and healthy controls and 380 clones differentially expressed between TB and sarcoidosis. Fig. 4 shows a Venn diagram depicting the overlap between 259 sarcoidosis markers, 238 TB vs. control and 380 TB vs. sarcoidosis markers. Clearly, 47 clones differentiate both sarcoidosis and TB from healthy controls, while 5 of them cannot differentiate sarcoidosis from TB. From these clones, 164 were found to be TB specific, and different from both healthy controls and sarcoidosis clones. Fig. 5 shows the heatmap of 50 significant clones differentially expressed in all three groups. Similarly to the sarcoidosis antigens, we have analyzed the specificity and sensitivity of TB clones to predict the presence of TB (Table 4). Finally, we sequenced 10 TB antigens, and sequence homologies were searched using the same algorithm as previously described. Table 4 shows the 10 TB-specific antigens as compared to healthy controls as well as sarcoidosis. After sequence analysis and homology search, we found one identical sequence between TB and sarcoidosis clone. Although the identified clone's name was different: P51_BP3_287 versus P51_BP3_174, and they performed differently in sarcoidosis versus TB as indicated in q value (compare Tables 3 and 4). However, using NCBI blast databases (mycobacterium toxoid and the universal blast) on the same sequence, two different proteins could be identified. Supplementary Table 2 shows the full length of protein and genes of 10 TB antigens. Surprisingly, TB clones show much higher sensitivity and specificity; similarly the AUROC was larger for the majority of TB antigens (Table 4).


Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens.

Talwar H, Rosati R, Li J, Kissner D, Ghosh S, -Madrid FF, Samavati L - EBioMedicine (2015)

Venn diagram depicts differential phage clone significances among sarcoidosis, TB and healthy controls (q < 0.01). Venn diagram shows the overlap between 259 sarcoidosis clones and 238 TB clones as compared to healthy controls, as well as 380 TB clones versus sarcoidosis. Forty seven clones could differentiate both sarcoidosis and TB from healthy controls. Five clones could not discriminate between TB and sarcoidosis.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465182&req=5

f0020: Venn diagram depicts differential phage clone significances among sarcoidosis, TB and healthy controls (q < 0.01). Venn diagram shows the overlap between 259 sarcoidosis clones and 238 TB clones as compared to healthy controls, as well as 380 TB clones versus sarcoidosis. Forty seven clones could differentiate both sarcoidosis and TB from healthy controls. Five clones could not discriminate between TB and sarcoidosis.
Mentions: In view of the clinical and pathological similarities between MTB and sarcoidosis, a most useful clinical antigen(s) should discriminate between these two conditions. To this end, using the antigens identified by biopanning the CSL library, a microarray was constructed and then this construct was interrogated with sera from 17 culture positive MTB subjects. Using a moderate t-test and a q value of < 0.05 in this system, we identified 238 clones differentially expressed between TB and healthy controls and 380 clones differentially expressed between TB and sarcoidosis. Fig. 4 shows a Venn diagram depicting the overlap between 259 sarcoidosis markers, 238 TB vs. control and 380 TB vs. sarcoidosis markers. Clearly, 47 clones differentiate both sarcoidosis and TB from healthy controls, while 5 of them cannot differentiate sarcoidosis from TB. From these clones, 164 were found to be TB specific, and different from both healthy controls and sarcoidosis clones. Fig. 5 shows the heatmap of 50 significant clones differentially expressed in all three groups. Similarly to the sarcoidosis antigens, we have analyzed the specificity and sensitivity of TB clones to predict the presence of TB (Table 4). Finally, we sequenced 10 TB antigens, and sequence homologies were searched using the same algorithm as previously described. Table 4 shows the 10 TB-specific antigens as compared to healthy controls as well as sarcoidosis. After sequence analysis and homology search, we found one identical sequence between TB and sarcoidosis clone. Although the identified clone's name was different: P51_BP3_287 versus P51_BP3_174, and they performed differently in sarcoidosis versus TB as indicated in q value (compare Tables 3 and 4). However, using NCBI blast databases (mycobacterium toxoid and the universal blast) on the same sequence, two different proteins could be identified. Supplementary Table 2 shows the full length of protein and genes of 10 TB antigens. Surprisingly, TB clones show much higher sensitivity and specificity; similarly the AUROC was larger for the majority of TB antigens (Table 4).

Bottom Line: Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients.Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls.These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Wayne State University School of Medicine and Detroit Medical Center, Detroit, MI 48201.

ABSTRACT

Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB). No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

No MeSH data available.


Related in: MedlinePlus