Secreted Antigens of Mycobacterium tuberculosis: Characterization with T Lymphocytes from Patients and Contacts after Two-Dimensional Separation

Background: Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder affecting 6–7% of premenopausal women. Recent studies revealed that the immune system especially CD4+ T helper cells are important in the context PCOS. Proteome analysis of CD4+ T lymphocytes can provide valuable information regarding the biology of these cells in the context of PCOS. Objective: To investigate immune dysregulation in CD4+ T lymphocytes at the protein level in the context of PCOS using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Methods: In the present study, we applied two-dimensional gel electrophoresis / mass spectrometry to identify proteins differentially expressed by peripheral blood CD4+ T cells in ten PCOS women compared with ten healthy women. Western blot technique was used to confirm the identified proteins. Results: Despite the overall proteome similarities, there were significant differences in the expression of seven spots between two groups (P <0.05). Three proteins, namely phosphatidylethanolamine-binding protein 1, proteasome activator complex subunit 1 and triosephosphate isomerase 1 were successfully identified by Mass technique and confirmed by western blot. All characterized proteins were over-expressed in CD4+ T cells from patients compared to CD4+ T cells from controls (P <0.05). In-silico analysis suggested that the over-expressed proteins interact with other proteins involved in cellular metabolism especially glycolysis and ferroptosis pathway. Conclusion: These findings suggest that metabolic adjustments in CD4+ T lymphocytes, which is in favor of increased glycolysis and Th2 differentiation are important in the context of PCOS.

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Purification of 38kDa Antigen of Mycobacterium tuberculosis by Two Dimensional Preparative Electrophoresis

BIO-PROTOCOL ◽

10.21769/bioprotoc.312 ◽

2012 ◽

Vol 2 (24) ◽

Author(s):

Uma Ranganathan

Keyword(s):

Mycobacterium Tuberculosis ◽

Two Dimensional ◽

Preparative Electrophoresis

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Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj2970351 ◽

1994 ◽

Vol 297 (2) ◽

pp. 351-357 ◽

Cited By ~ 95

Author(s):

A Lemassu ◽

M Daffé

Keyword(s):

Mycobacterium Tuberculosis ◽

Molecular Mimicry ◽

Degradation Products ◽

Cell Envelope ◽

Gel Filtration ◽

Structural Features ◽

Two Dimensional ◽

Phagocytic Cells ◽

Molecular Masses ◽

Branched Structure

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

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Effect of Growth State on Transcription Levels of Genes Encoding Major Secreted Antigens of Mycobacterium tuberculosis in the Mouse Lung

Infection and Immunity ◽

10.1128/iai.72.4.2420-2424.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2420-2424 ◽

Cited By ~ 60

Author(s):

Lanbo Shi ◽

Robert North ◽

Maria Laura Gennaro

Keyword(s):

Mycobacterium Tuberculosis ◽

Adaptive Immunity ◽

Mouse Lung ◽

Mrna Levels ◽

Growth State ◽

Genes Encoding ◽

Secreted Antigens

ABSTRACT Arrest of the multiplication of Mycobacterium tuberculosis caused by expression of adaptive immunity in mouse lung was accompanied by a 10- to 20-fold decrease in levels of mRNAs encoding the secreted Ag85 complex and 38-kDa lipoprotein. esat-6 mRNA levels were high throughout infection. The data imply that multiplying and nonreplicating tubercle bacilli have different antigen compositions.

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Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822004000600005 ◽

2004 ◽

Vol 37 (6) ◽

pp. 454-459

Author(s):

Adriano Gomes Silva ◽

Elisangela Paula Silveira-Lacerda ◽

Jair Pereira Cunha-Júnior ◽

Maria Aparecida de Souza ◽

Silvio Favoreto Junior

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Gel Electrophoresis ◽

Complex Mixture ◽

Chronic Phase ◽

Disease Diagnosis ◽

Basic Knowledge ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Secreted Antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

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PERCENTAGE OF CD3+ T LYMPHOCYTES EXPRESSING IFN-γ AFTER CFP-10 STIMULATION (Persentase Limfosit T-CD3+ yang Mengekspresikan Interferon Gamma Setelah Stimulasi Antigen CFP-10)

INDONESIAN JOURNAL OF CLINICAL PATHOLOGY AND MEDICAL LABORATORY ◽

10.24293/ijcpml.v23i1.1181 ◽

2018 ◽

Vol 23 (1) ◽

pp. 31

Author(s):

Yulia Nadar Indrasari ◽

Betty Agustina Tambunan ◽

Jusak Nugraha ◽

Fransiska Sri Oetami

Keyword(s):

Flow Cytometry ◽

Mycobacterium Tuberculosis ◽

T Lymphocytes ◽

Interferon Gamma ◽

Tuberculosis Antigen ◽

Mycobacterium Tuberculosis Antigen ◽

Ifn Γ

Tuberkulosis (TB) merupakan penyakit infeksi menular, disebabkan oleh Mycobacterium tuberculosis. Respons imun adaptif yangdiperantarai oleh limfosit T berperan sangat penting dalam menyingkirkan bakteri intraseluler. Hasilan sitokin IFN-γ merupakanmekanisme efektor utama dari limfosit T. Pengembangan vaksin yang efektif dalam melawan infeksi TB mempertimbangkan faktor yangmengatur hasilan IFN-γ. CFP-10 merupakan antigen yang disekresikan oleh Mycobacterium tuberculosis. Antigen ini dikenal sebagaikomponen vaksin potensial untuk TB. Tujuan penelitian ini adalah membandingkan respons imun seluler yaitu persentase limfosit T-CD3+yang mengekspresikan IFN-γ setelah dirangsang antigen CFP-10 di pasien TB paru kasus baru, TB laten dan orang sehat. Penelitianini menggunakan desain eksperimen murni di laboratorium secara in vitro pada kultur PBMC pasien TB paru kasus baru, TB latendan orang sehat. Subjek penelitian adalah 8 pasien TB paru kasus baru, 7 TB laten dan 7 orang sehat di RS Khusus Paru Surabaya.Pemeriksaan persentase limfosit T-CD3+ yang mengekspresikan IFN-γ dengan metode Flow cytometry (BD FACSCalibur). Hasil dianalisisdengan Kruskal-Wallis atau ANOVA satu arah. Rerata persentase limfosit T-CD3+ yang mengekspresikan IFN-γ di TB paru kasus barusetelah stimulasi antigen CFP-10 (4,36%) lebih tinggi daripada sebelum stimulasi (3,50%) (nilai P=0,015). Rerata persentase limfositT-CD3+ yang mengekspresikan IFN-γ di TB laten setelah stimulasi antigen CFP-10 (3,96%) lebih tinggi dibandingkan sebelum stimulasi(2,50%) tetapi tidak bermakna (nilai P=0,367). Rerata persentase limfosit T- CD3+ yang mengekspresikan IFN-γ di orang sehat setelahstimulasi (1,66%) lebih rendah daripada sebelum stimulasi (2,89%) tetapi tidak bermakna (nilai P=0,199). Perubahan persentaselimfosit T-CD3+ yang mengekspresikan IFN-γ setelah stimulasi antigen CFP-10 antarkelompok tidak berbeda bermakna (nilai P=0,143).Berdasarkan hasil telitian ini dapat disimpulkan bahwa terdapat peningkatan persentase limfosit T-CD3+ yang mengekspresikan IFN-γdi TB paru kasus baru setelah stimulasi antigen CFP-10. Hal ini menunjukkan limfosit T-CD3+ yang mengekspresikan IFN-γ berperandalam perlindungan terhadap infeksi TB paru.

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