T lymphocyte subsets combined with inflammatory indicators for auxiliary diagnosis and prognosis evaluation of sepsis / 中华预防医学杂志

Objective: To explore the application value of T lymphocyte subsets combined with procalcitonin (PCT), C-reactive protein (CRP), neutrophil to lymphocyte ratio (NLR) and white blood cell count (WBC) in the auxiliary diagnosis and prognosis evaluation of sepsis. Methods: In a retrospective study, seventy-two patients with sepsis diagnosed and treated in Tianjin First Central Hospital from June 2018 to April 2021 were selected as the research objects, and included in the sepsis group were 46 males and 26 females, aged 68 (57.3, 80.3) years. In addition, 111 patients with local infection admitted to hospital during the same period were included in the local infection group, including 62 males and 49 females, aged 68 (51, 77) years. Sepsis patients were divided into survival group (43 cases) and death group (29 cases) according to the 28-day outcome. CD3+, CD4+, CD8+, CD4+/CD8+ ratio were detected by flow cytometry within 24 h after admission, PCT was detected by ELISA, CRP was detected by immunoturbidimetry, blood routine examination, blood lactic acid (Lac) and oxygen partial pressure (PO2) were detected by instrumental method. Multivariate Logistic regression analysis was used to evaluate the correlation between each indicator and sepsis, and receiver operating characteristic curve (ROC) was drawn to evaluate the diagnostic value of each indicator for sepsis. Multivariate Logistic regression analysis and Kaplan Meier survival analysis were used to evaluate the prognostic value of each index for patients with sepsis. Results: Peripheral blood CD3+, CD4+, CD8+, CD4+/CD8+ ratio and PLT in sepsis group were significantly lower than those in local infection group(Z=-8.184,P<0.001;Z=-7.210,P<0.001;Z=-5.936,P<0.001;Z=-2.700,P=0.007;Z=-6.381,P<0.001); PCT, CRP, NLR and Lac levels were significantly higher than those in local infection group(Z=-8.262,P<0.001;Z=-3.094,P=0.002;Z=-9.004,P<0.001;Z=-4.770,P<0.001). Multivariate Logistic regression model showed that PCT, NLR, CD3+, CD8+, CD4+/CD8+ were independent risk factors for sepsis. According to ROC curve analysis, AUC of sepsis patients diagnosed by each indicator were 0.862, 0.894, 0.858, 0.760 and 0.618, respectively. The cut-off values were 3.075 ng/ml, 10.715, 44.935×109/L, 27.463×109/L and 0.750, respectively. The NLR sensitivity was 80.6%, and the CD3+ specificity was 94.6%. The AUC of combined detection of PCT and NLR was 0.947, sensitivity was 87.5% and specificity was 91.9%. The combined detection AUC of PCT, NLR, CD3+, CD4+/CD8+ was 0.958, the sensitivity and specificity were 90.3% and 91.0% respectively(P<0.001). PCT and Lac in death group were significantly higher than those in survival group(Z=-2.302,P=0.021;Z=-3.095,P=0.002);Peripheral blood CD4+/CD8+ levels were significantly lower than those in survival group(Z=-3.691,P<0.001),Multivariate Logistic regression model showed that CD4+/CD8+ ratio was an independent risk factor for 28 d mortality in patients with sepsis (P<0.001). The ROC curve showed that the AUC was 0.758, and the Youden index reached the maximum when the cut-off value was 1.27, the sensitivity and specificity were 79.3% and 60.5%, respectively. Compared with patients with CD4+/CD8+ ≥1.27, 28-day mortality was significantly increased in patients with CD4+/CD8+<1.27 (P=0.032). Conclusion: The combined detection of PCT, NLR, CD3+ and CD4+/CD8+ can improve the auxiliary diagnostic efficiency of sepsis, and the ratio of CD4+/CD8+ in peripheral blood may have certain predictive value for the prognosis of sepsis.

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo / Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFN ? , IL-2 y TNFa frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi . Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFN ? , IL-2 y TNFa fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi . Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica.

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.

Serum cytokine levels in autoimmune and non-autoimmune hyperthyroid states

Although the role of interleukin-2 (IL-2) and interferon gamma (yIFN) is still poorly understood in hyperthyroid diseases, it is reasonable to assume that these cytokines may be present at higher levels in Graves' disease (GD) than in other primarily non-autoimmune thyroid diseases. In order to look for an easy method to distinguish GD from primarily non-autoimmune causes of hyperthyroidism, we compared 13 healthy individuals with 21 treated and untreated hyperthyroid GD patients and with 19 patients with hyperthyroidism due to other etiologies: 7 cases of multinodular goiter, 5 cases of excessive hormone replacement and 7 cases of amiodarone-associated hyperthyroidism. All patients presented low TSH levels and a dubious clinical thyroid state. We found a good correlation between TSH and serum IL-2 levels (r = 0.56; P

Immunological alterations in patients with primary tumors in central nervous system

Natural Killer (NK) cells play an important role in immune surveillance against tumors. The present work aimed to study the cytotoxic activity of NK cells and T cell subsets in peripheral blood of 13 patients with primary in central nervous system (CNS). As controls 29 healthy subjects with the age range equivalent to the patients were studied. The methods employed were: a) determination of cytotoxic activity of NK cells towards K562 target cells, evaluated by single cell-assay; b) enumeration of CD3+ lymphocytes and their CD4+ and CD8+ subsets defined by monoclonal antibodies; c) the identification of tumors were done by histologic and immunochemistry studies. The results indicated that adults and children with tumor in CNS display reduced percentage of total T cells, helper/inducer subset and low helper/suppressor ratio. The cutotoxic activity of NK cells was decreased in patients with CNS tumors due mainly to a decrease in the proportion of target-binding lymphocytes. These results suggest that cytotoxic activity of NK cells may be affected by the immunoregulatory disturbances observed in patients with primary tumors in CNS.

The cellular immune status of HBsAg-positive carriers in Malaysia.

The percentage of lymphocyte subsets from the peripheral blood of healthy adults and hepatitis B surface antigen (HBsAg) carriers were analyzed by flow cytometry. The five lymphocyte subsets studied were:- T (CD3) cells, B (CD19) cells, CD4 cells, CD8 cells, Natural Killer (CD3- CD16+/CD56+) cells (NK cells) and the CD4/CD8 ratio. The percentage (mean +/- SD) for the five lymphocyte subsets from the healthy adults were (67.5 +/- 8.5)%, (12.4 +/- 4.5)%, (35.5 +/- 7.8)%, (36.8 +/- 8.5)%, (17.9 +/- 8.1)% and 1.1 +/- 0.6, respectively. HBsAg carriers positive for HBV-DNA had a lower CD4/CD8 ratio than the healthy population (P = 0.030). The percentage of CD8 cells in HBsAg carriers increased significantly (r = 0.28; P = 0.019) with an increase in ALT levels but the values remained within normal range. The percentage of NK cells and CD4/CD8 ratio in HBsAg carriers positive for anti-HBe were higher than HBsAg carriers negative for anti-HBe (92% of which are HBeAg positive) (P = 0.045 and P = 0.035, respectively). The CD4/CD8 ratio in HBsAg carriers negative for anti-HBe (92% positive for HBeAg) was also lower than in the healthy population (P = 0.042). HBsAg carriers positive for HBV-DNA, HBeAg and raised ALT levels had a lower CD4/ CD8 ratio than did the healthy population. The lower ratio was due to an increase in the percentage of CD8 cells. This suggests an activated immune response triggered by the infection in an attempt to clear the virus. HBsAg carriers with normal ALT levels and who are negative for HBV-DNA may be in a state of tolerance.

Evaluacion de las subpoblaciones de linfocitos T en pacientes tuberculosos empleando la modulacion con teofilina / T-lymphocyte subsets evaluation in patients with tuberculosis using modulation with theophylline

Se evaluaron las poblaciones y subpoblaciones linfocitarias en pacientes con tuberculosis pulmonar antes y durante la terapia relacionando estos valores con la incidencia y evolucion de la enfermedad. Pacientes en sus diversas manifestaciones clinicas, virgenes de tratamiento, se estudiaron por baciloscopia (BAAR), radiologia, i.d.r. Mantoux y analisis complementarios. Se cuantificaron mediante la prueba de Rosetas espontaneas (RE) linfocitos T totales y activos (RE a 4 graus Celsius y 37 graus Celsius), T colaboradores (RE Teofilina Resistentes: RETR) y supressores (RE Teofilina Sensibles: RETS). Los examenes se repitieron en los mismos sujetos iniciado el tratamiento y en testigos sanos (TS). Se demostro en los pacientes en todas sus formas clinicas un descenso significativo en los valores relativos y absolutos de celulas T y en la relacion RETR/RETS (menor de 1). Existe asociacion entre la forma clinica y el numero de linfocitos T colaboradores. Los pacientes en tratamiento con evolucion favorable, evidenciaron un incremento significativo en los linfocitos T totales, activos, colaboradores y en la relacion RETR/RETS. Los enfermos con baciloscopia altemente positiva presentaron i.d.r. Mantoux baja o negativa y marcado descenso de celulas inmunocompetentes...

Defectos inmunológicos en el shock séptico: deficiencia de células natural killer y de linfocitos T / Immunological defects in septic shock: deficiency of natural killer cells and T-lymphocytes

It is well fnown that an immunosuppresive rsponse occuts after acute trauma. Some cellular mediators participate in the pathogenesis of septic shock. However, the exact role of the lymphocyte subsets and natural killer (NK) activity in this condition is not clear. We studied NK cytolytic activity through a 51Cr liberation assay using K-562 target cells in 20 patients with initial septic shock (10 men and 10 females, mean age 41 years old). Lymphocyte subsets CD3 (T3), CD4 (T4), CD8 (T8), CD16 (Leu-11) and CD56 (Leu-19) wetre also studied by indirect immunofluorescence. Compared to tesults obtained in 20 healthy volunteers, patient's NK activity was decreased (4.6 ñ 3.9 vs 26.1 ñ 10, p < 0.025), CD16 was lower (10%/187 vs 15%/280 per ul) and CD56 was also lower (6%/120 vs 12%/224 per ul), p < 0,05. T lymphocyte subsers were also decreased: CD3 cells (1100 vs 1352 per ul) and CD4 cells (634 vs 873 per ul), p < 0.05. Thus, a severe decrease in NK cells and NK cell function as well as decreases in CD3 and CD4 lymphocyte subsets are present in the initial stages of septic shock. The predictive value of these findings is currently under study

The association of adenosine deaminase activity with T-lymphocytes and subsets in pulmonary tuberculosis and bronchogenic carcinoma.

Simultaneous determination of blood/lung ADA activity and T-lymphocyte subsets was conducted in 12 patients with active pulmonary tuberculosis, 12 patients with bronchogenic carcinoma and 11 healthy volunteers. Differences were significant only in the tuberculosis patients, namely, increased mean enzyme values in both the peripheral blood (36.68 +/- 10.90 U/L) and in the BALF (4.25 +/- 2.19 U/L), and correlation of ADA activity between the blood and the diseased lung only; the difference in elevated enzymatic activity between the tuberculosis group and the cancer group was of no statistical significance. We conclude that simultaneous ADA analysis of the blood and the BALF may be of diagnostic value in cases suspected of having tuberculosis as yet undiagnosed by other means. Based on the lowest value of enzymatic activity in the blood of patients with tuberculosis (28 U/L), the test has a sensitivity of 75 per cent and a specificity of 100 per cent; whereas the lowest value in the BALF of tuberculosis patients (2.9 U/L), the test has a sensitivity of 77 per cent and a specificity of 82 per cent. Findings that there was a blood-lung correlation of elevated ADA activity and a correlation of enzymatic elevation with increased numbers of T-cells bearing IL-2 receptor in cases of pulmonary tuberculosis only provide evidence in support of T-lymphocytes actively participating in the ongoing immune process.