To investigate the relationship between 9 serum cytokines and clinical, subclinical characteristics in patients with new AFB negative pulmonary tuberculosis. 88 patients with AFB-negative pulmonary tuberculosis were treated at Department of Tuberculosis and Lungs Diseases, 198 Hospital.
Journal of military pharmaco-medicine no7-2018 RESULTS OF SERUM CYTOKINES IN PATIENTS WITH NEW AFB NEGATIVE PUMONARY TUBERCULOSIS Dinh Thi Hoa*; Tran Van Sau*; Nguyen Huy Luc** SUMMARY Objectives: To investigate the relationship between serum cytokines and clinical, subclinical characteristics in patients with new AFB negative pulmonary tuberculosis Subjects and methods: 88 patients with AFB-negative pulmonary tuberculosis were treated at Department of Tuberculosis and Lungs Diseases, 198 Hospital We performed nine simultaneous cytokines tests (including IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN gamma, TNF alpha) at Immuno Labo-Center for Medicine and Pharmacy, Military Medical University Results: Mean concentrations of cytokines: IL-4: 1.41; IL-5: 6.96 ± 5.47; IL-10: 90.11 ± 359.32; IL-12: 79.65 ± 292 47; IL-13: 5.49 ± 13.81; TNF: 285.6 ± 1932.36, alpha TNF: 12.57 ± 13.16 The group of weight loss patients had a decreased cytokine value compared with the unweight group, with a difference of p < 0.05 In patients with fever, mean values of some cytokines were lower than those without fever, difference was significant (p < 0.05) Conclusion: There was a relation between serum cytokines levels and some clinical, subclinical characteristics in new AFB negative tuberculosis patients * Keywords: New AFB negative pulmonary tuberculosis; Cytokines INTRODUCTION Cytokines are proteins with a low molecular weight, which mediates signal exchange between cells of the immune system Cytokines work by binding to specific receptors on the target cell, thereby activating signaling pathways within the cell that alter the expression of the gene resulting in the synthesis of new protein determines the biological effect So far, two subtypes of TH cells, TH1 and TH2, have been known to date and the types of immune responses they produce Some tuberculosis (TB)-related cytokines are also produced in this mechanism and have particular implications for the pathogenesis and progression of the disease They are cytokines: IL-2, IL-4, IL-5, IL-10, IL-12 and IL-13, interferon gamma, TNF alpha, CSF We conducted this study aimed at: Simultaneous detection of cytokines: IL-2, IL-4, IL-5, IL-10, IL-12 and IL-13, interferon gamma, TNF alpha, CSF serum and associated with clinical and laboratory symptoms in patients with AFB-negative pulmonary TB * 198 Hospital ** 103 Military Hospital Corresponding author: Dinh Thi Hoa (hoadinh198@gmail.com) Date received: 27/06/2018 Date accepted: 24/08/2018 139 Journal of military pharmaco-medicine no7-2018 SUBJECTS AND METHODS Subjects 88 patients who were diagnosed with AFB negative pulmonary tuberculosis (TB) were taken blood at the time of admission Right after centrifugation blood plasma was separated and then divided into 1.5 mL of tubes and kept at 70 degrees negative until the test Methods - The quantification of cytokines was detected by Sandwich-type immunoassay on the surface of micro plastic particles Based on the fluorescence densities derived from the incubated seeds with known cytokine concentrations that allow the cytokine to be quantified - Determination of association of cytokine concentrations with clinical and subclinical symptoms + Based on the quantitative concentration of cytokines and then divided into groups of clinical and subclinical clinical symptoms RESULTS Mean concentrations of serum cytokines (pg/mL) Table 1: Cytokine Medium Min - max IL-2 9.62 ± 55.33 0.53 - 506.46 IL-4 1.59 ± 1.41 0.25 - 9.12 IL-5 6.96 ± 5.47 1.70 - 31.42 IL-10 90.11 ± 359.32 2.13 - 2752.94 IL-12 79.65 ± 292.47 0.02 - 1742.69 IL-13 5.49 ± 13.81 0.40 - 129.98 IFN gamma 285.67 ± 1932.36 12.18 - 1904.18 TNF alpha 13.57 ± 13.16 2.41 - 85.90 CSF 67.36 ± 75.58 1.41 - 380.93 Relations between cytokine levels and clinical and subclinical symptoms Table 2: Relations between the concentration of cytokines and symptoms of weight loss Cytokine No weight loss (n = 48) Weight loss (n = 40) p IL-2 15.89 ± 74.59 2.09 ± 4.04 0.269 IL-4 1.89 ± 1.67 1.24 ± 0.91 0.032 IL-5 8.12 ± 6.49 5.55 ± 3.51 0.075 IL-10 136.68 ± 469.23 34.22 ± 129.63 0.011 IL-12 114.71 ± 363.61 37.58 ± 167.75 0.009 140 Journal of military pharmaco-medicine no7-2018 IL-13 7.37 ± 18.45 3.24 ± 2.36 0.013 IFN gamma 369.12 ± 422.87 185.54 ± 245.26 0.021 TNF alpha 16.36 ± 15.94 10.22 ± 7.67 0.062 CSF 86.88 ± 84.91 43.94 ± 54.99 0.005 Results of the above table showed that the weight loss group had significantly lower cytokine values than those without weight loss, significant difference with p < 0.05 (IL-4, IL-10, IL-12, IL-13, IFN gamma and CSF) Table 3: Relations between cytokine levels and fever symptoms Cytokine No fever (n = 48) Fever (n = 40) p IL-2 15.39 ± 74.67 2.69 ± 4.33 0.927 IL-4 1.97 ± 1.73 1.15 ± 0.66 0.015 IL5 8.27 ± 6.65 5.38 ± 2.97 0.047 IL-10 80.19 ± 267.42 101.99 ± 448.96 0.069 IL-12 91.32 ± 329.11 65.64 ± 244.74 0.024 IL-13 5.05 ± 3.89 6.01 ± 20.17 0.003 IFN gamma 377.59 ± 446.01 175.36 ± 178.59 0.054 TNF alpha 16.86 ± 16.29 9.62 ± 6.07 0.033 CSF 80.65 ± 81.16 51.42 ± 65.79 0.044 Patients with fever, mean values of some cytokines were significantly lower than those without fever, the difference was statistically significant at p < 0.05 (IL-4, IL-5, IL-12, IL-13, CSF and TNF alpha) Table 4: Relations between cytokine levels and functional symptoms Cytokine Dry cough (n = 62) Blood cough (n = 18) Cough with sputum (n = 8) (p) IL-2 12.53 ± 65.79 2.71 ± 4.74 2.63 ± 3.89 0.969 IL-4 1.47 ± 1.18 2.34 ± 2.04 0.84 ± 0.56 0.011 IL-5 6.44 ± 4.97 9.42 ± 7.11 5.38 ± 3.64 0.089 lL-10 118.58 ± 425.61 26.79 ± 24.93 11.87 ± 10.76 0.083 IL-12 103.59 ± 345.99 24.46 ± 26.45 18.21 ± 29.84 0.221 IL-13 5.75 ± 16.29 5.60 ± 4.15 3.22 ± 3.05 0.049 IFN gamma 257.27 ± 319.78 482.27 ± 489.88 63.46 ± 62.84 0.003 TNF alpha 12.30 ± 10.92 20.67 ± 19.23 7.43 ± 5.26 0.014 CSF 56.38 ± 61.09 95.04 ± 95.90 90.23 ± 111.88 0.234 Patients with coughing sputum tend to have lower mean concentrations of cytokines than those with dry coughs and coughs, however, the difference was statistically significant only for IL-4; IL-13, IFN gamma and TNF alpha 141 Journal of military pharmaco-medicine no7-2018 Table 5: Relations between lesion on chest X-ray and cytokine concentration Lesions Large (n = 19) Medium (n=34) Narrow (n = 35) p IL-2 1.32 ± 2.41 17.92 ± 86.47 6.07 ± 21.27 0.109 IL-4 1.36 ± 0.75 1.56 ± 1.43 1.76 ± 1.66 0.710 IL-5 6.39 ± 3.33 7.05 ± 6.05 7.17 ± 5.91 0.942 IL-10 14.59 ± 9.0 199.95 ± 564.70 24.39 ± 33.50 0.509 IL-12 12.76 ± 10.38 178.80 ± 456.43 19.64 ± 23.79 0.578 IL-13 3.21 ± 1.72 7.84 ± 21.89 4.44 ± 3.45 0.548 IFN gamma 202.48 ± 213.12 308.08 ± 382.61 309.06 ± 408.62 0.654 TNF alpha 11.04 ± 7.16 12.88 ± 13.28 15.62 ± 15.36 0.299 CSF 44.66 ± 38.99 68.05 ± 70.13 79.03 ± 92.80 0.732 Cytokine The difference was not statistically significant between the mean concentrations of cytokines and the extent of injury (p > 0.05) Table 6: Relations between serum cytokine levels and peripheral white blood cell characteristics of the two groups Cytokine White blood cells normal (n = 73) White blood cells increase (n = 15) p IL-2 11.19 ± 60.66 1.96 ± 4.76 0.254 IL-4 1.62 ± 1.51 1.48 ± 0.77 0.549 IL-5 6.77 ± 5.20 7.84 ± 6.76 0.387 IL-10 93.95 ± 383.83 71.37 ± 210.23 0.272 IL-12 78.56 ± 298.14 84.95 ± 272.66 0.542 IL-13 5.77 ± 15.12 4.13 ± 2.78 0.637 IFN gamma 293.25 ± 385.63 248.79 ± 232.12 0.609 TNF alpha 13.76 ± 14.08 12.67 ± 7.37 0.474 CSF 70.08 ± 79.62 54.17 ± 51.76 0.789 The table above shows that cytokine concentrations were not associated with elevated white blood cell counts (p > 0.05) DISCUSSION Average concentration of serum cytokines For years, authors in many countries around the world have focused on the issue of immunity in TB In terms of the 142 occurrence of cytokines, and the increase in the concentration in each stage of the disease has been analyzed and described, each cytokine specific to the pathology as well as the relations to the clinical and laboratory findings are statistically significant Journal of military pharmaco-medicine no7-2018 However, in order to obtain a standardized outcome and to diagnose definite TB disease, it is not yet clear, so all the studies are only at the level of reference and continue to provide evidence of relevant value [1, 2, 3] Our study also only commented on serum cytokine levels in patients with AFB-negative pulmonary TB and their association with some subclinical indications TB immunity is a cell-mediated immune response through T-lymphocytes and secreted cytokines in which the cytokine plays an important role, such as gamma IFN, IL-12, alpha TNF, IL-2, IL-4, IL-5, IL-10, IL-13 and GM-CSF [6] Wu H et al (2007) found that in non-TB patients, the level of IFN gamma was higher in patients with pulmonary TB disease [4] Sahiratmadja E et al (2007) revealed that the concentration of IFN gamma decreased dramatically in untreated TB He argues that IL-10 inhibits the generation of IFN gamma by reducing IL-12 receptor expression leading to a decrease in IL-12 response and inhibition of Th1 immune response It also indicates that alpha TNF increases in active TB and correlates with the level of activity of the disease: increases without treatment and decreases when the disease is stabilized [5] Ribeiro Rodrigues R et al (2006) found that serum IL-10 levels increased and returned to normal level after months of treatment, while gamma IFN decreased when treatment was complete In our study, the mean values of cytokines in the serum of patients with new AFB-negative pulmonary TB were at baseline Relations between serum cytokine levels and some clinical and subclinical symptoms Verbon A et al in 1999 found that gamma IFN increased markedly in patients with fever compared to patients without fever and this cytokine level was not associated with weight loss [9] Kart I.L (2003), Berker L.G et al (1998), Buyuckoglan H (2007) showed elevated alpha TNF levels in patients with systemic symptoms such as persistent fever, weight loss, but the difference was statistically significant [10] Our research is well suited to these authors: - Patients with weight loss had significantly lower cytokine values than those without weight loss - The difference was statistically significant with p < 0.05 (IL-4, IL-10, IL-12, IL-13, gamma IFN and CSF) - Patients with fever, the median mean of some cytokines was lower than that of the non-fever group, the difference was statistically significant at p < 0.05 (IL-4, IL-5, IL-12, IL-13, CSF and TNF alpha) - Patients with cough with sputum usually had lower mean concentrations of cytokines than those with dry cough and cough, however, the difference was statistically significant only for IL-4; IL-13, gamma IFN and TNF alpha Some authors suggested that the concentration of some cytokines were related to the lesion area on the X-ray and the extent of destruction of the cervical crest, as well as to the blood cell and white blood cell indices, difference was not statistically significant [8] 143 Journal of military pharmaco-medicine no7-2018 In our study, no correlation was found between cytokine levels and these subclinical symptoms detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells Clin Diagn Lab Immunol 2003, 10 (1), pp.133-139 CONCLUSION Goldsby R.A, Kindt T.J, Osborne B.A, th Kuby J Cytokines in: Kuby Immunology Ed” Freeman and Company 2003, pp.276-298 - Average concentration of cytokines in the blood: IL-2: 9.62 ± 55.33; IL-4: 1.41; IL-5: 6.96 ± 5.47; IL-10: 90.11 ± 359.32; IL-12: 79.65 ± 292.47; IL-13: 5.49 ± 13.81; INF: 285.6 ± 1932.36; alpha TNF: 12.57 ± 13.16 The group of weight loss patients had significantly lower values of cytokine compared with those who did not lose weight The differences were statistically significant at p < 0.05 (IL-4, IL-10, IL-12, IL-13, IFN gamma and CSF) Patients with fever, mean values of some cytokines lower than those without fever, the difference was statistically significant at p < 0.05 (IL-4, IL-5, IL-12, IL-13, CSF and TNF alpha) - Patients with coughing sputum tend to have lower mean concentrations of cytokines than those with dry cough, however, the difference was statistically significant only for IL-4; IL-13, TNF gamma and TNF alpha REFERENCES Hoang Thi Phuong, Tran Van Sang, Ho Minh Ly, Tran Thi Thanh Hoa Disease detection time and some immunological features in patients with pulmonary TB and non-diabetic patients with pulmonary TB Annual Meeting of Immunology Teaching and Research 2007, pp.51-54 De Jager W, Te Velthuis H, Prakken B.J, Kuis W, Rijkers G.T Simultaneous 144 Wu H.P, Hua C.C, Liu Y.C et al Comparison of interferon-gamma response between TB and non–TB pneumonia Inflamm Res 2007, 56, pp.11-16 Sahiratmadja E, Alisjahbana B, Boer T et al Dynamic changes in pro-and antiinflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with TB disease activity and response to curative treatment Infection and Immunity 2007, 75 (2), pp.820-829 Moura E.P, Toledo V.P C.P, Oliveira M.H.P et al Pulmonary TB: Evaluation of interferon gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Gurein Mem Inst Oswaldo Cruz 2004, 99 (3) Deveci F, Akbulut H.H, Turgut T et al Changes of serum cytokine levels in active TB with treatment Mediator of Inflammation 2005, 5, pp.256-262 Wu H.P, Hua C.C, Chuang D.Y Decreased in vitro interferon-gamma production in patients with cavitary TB on chest radiography Respir Med 2004 Verbon A.L, Juffemans N, Van Deventer S.L Serum concentration of cytokines in patients with active TB and after treatment Clin Exp Immunol 1999, Jan, 115 (1), pp.110-113 10 Kart I.L, Buyukoglin H, Tekin I.O et al Correlation of serum TNF anpha, Il-4 and soluble IL-2, receptor levels with radiologic and clinical manifestation in active pulmonary TB Mediator of Inflammation 2003, 12, pp.9-14 ... concentration of cytokines and then divided into groups of clinical and subclinical clinical symptoms RESULTS Mean concentrations of serum cytokines (pg/mL) Table 1: Cytokine Medium Min - max IL-2 9. 62... in the serum of patients with new AFB- negative pulmonary TB were at baseline Relations between serum cytokine levels and some clinical and subclinical symptoms Verbon A et al in 199 9 found that... S.L Serum concentration of cytokines in patients with active TB and after treatment Clin Exp Immunol 199 9, Jan, 115 (1), pp.110-113 10 Kart I.L, Buyukoglin H, Tekin I.O et al Correlation of serum