Determination of reference values for TREC and KREC in circulating blood of the persons over 18 years

Increasing attention is being paid to methods for detecting primary and secondary T and/or B cell immunodeficiencies. Their implementation into laboratory diagnostics would contribute to the early diagnostics of immunodeficiencies. Currently, the number of identified adult patients with immunodeficiencies of various origins is steadily increasing. Age, gender and ethnicity of patients may be significant factors of immunity. Hence, determination of the population reference intervals for TREC and KREC DNA excision rings in peripheral blood of adult persons is an urgent laboratory task for in-depth examination of both congenital and acquired immunodeficiency conditions. Our purpose was to determine the reference intervals for the quantitative assay of TREC and KREC fragments in peripheral blood among the adult population of St. Petersburg. We studied whole blood samples obtained from 717 apparently healthy volunteers aged 18 to 108 years within the program of population immunity assessment among residents of St. Petersburg. The exclusion criterion included immunodeficiency of any origin, viral hepatitis A, B, C, HIV infection. Quantitation of the target TREC and KREC DNA fragments was carried out using a set of reagents for the quantitative determination of excisional rings TREC and KREC by Real-time PCR (TREC/KREC-AMP PS). The reference intervals were determined by the direct method according to the recommendations of the International Federation of Clinical Chemistry and the Russian State Standard (GOST) R 53022.3-2008. The volunteers were divided into six age groups: 18-29, 30-39, 40-49, 50-59, 60-69 years old, and the persons over 70. The amounts of TREC and KREC in each blood sample were determined for all age groups. Upon correlation analysis, we have revealed a negative relationship between the concentration of TREC molecules in blood samples, and the age of study participants (Spearman correlation coefficient r = -0.80 (p-value < 0.0001)). Significant differences in TREC levels between different age groups were revealed. No correlations were detected between KREC contents in blood samples and age as well as any differences between age groups. Reference intervals of the TREC level were determined for each mentioned age group. A unified reference range was established for the KREC levels. The established reference intervals for TREC and KREC molecules in adults are significantly lower than in newborns. The obtained results enable determination of reference intervals for TREC and KREC levels among adults, thus contributing to effective personalized laboratory diagnosis of immunodeficiency states of various origins

Features of cytokine profile in patients with sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology with damage to the lungs and other organs characterized by development of necrosis-free epithelioid cell granulomas. Granulomatous inflammation characterized by the activation of different immune systems cells, in particular T lymphocytes, and the cytokines production. Our study was aimed at investigating the characteristics of the cytokine profile of blood plasma in patients with sarcoidosis. We studied peripheral blood plasma samples of patients with sarcoidosis (n = 52). The control blood samples were taken from healthy volunteers (n = 22). The level of 46 cytokines (pg/ml) was determined, as follows: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IFNα2, IFNγ, TNFα, TNFβ, IL- 1ra, IL-10, EGF, FGF-2, Flt3 Ligand, G-CSF, GM-CSF, PDGF-AA, PDGF-AB / BB, TGFα, VEGF-A, sCD40L, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL17, CCL20, CCL22, CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1. Significantly higher levels of interleukins and some proinflammatory cytokines were found in the patients with sarcoidosis, i.e., IL-3, 0.70 vs 0.20, p = 0.003; IL-4, 14.37 vs 3.15, p = 0.009; IL-5, 1.06 vs 0.89, p < 0.001; IL-12 (p70), 1.27 vs 0.56, p = 0.028; IL-17A, 1.48 vs 0.43, p < 0.001; IFNα2, 41.79 vs 25.04, p = 0.003; IFNγ, 4.13 vs 1.14, p < 0.001; TNFα, 21.67 vs 6.70, p < 0.001; anti-inflammatory cytokine IL-10, 1.03 vs 0.45, p = 0.019; growth factors: FGF-2, 40.08 vs 30.58, p = 0.008, G-CSF, 24.18 vs 8.21, p = 0.006, and VEGF-A, 42.52 vs 26.76, p = 0.048; chemokines: CCL3, 3.86 vs 1.33, p < 0,001; CCL17, 78.24 vs 26.24, p < 0.001; CCL20, 7.19 vs 5.64, p = 0.021; CCL22, 660.60 vs 405.00, p < 0,001; CXCL9, 4013 vs 1142, p < 0,001; CXCL10, 565.90 vs 196.60, p < 0.001; CXCL11, 230.20 vs 121.10, p = 0.018; CX3CL1, 56.99 vs 5.16, p < 0.001. Peripheral blood chemokine CCL11 levels were significantly lower in patients compared to the group of healthy volunteers: 77.58 vs 124.70, p = 0.022. The features of the cytokine profile in patients with sarcoidosis may indicate their important role in the processes of formation and outcomes of granulomas. These issues require an additional detailed study, comparison with phenotypes, differential course and outcomes of the disease

CXCR3 chemokine receptor ligands in sarcoidosis

Sarcoidosis is a polysystemic inflammatory disease of unknown etiology, morphologically related to the group of granulomatosis, with heterogeneous clinical manifestations and outcomes. Immune cells, in particular T helper cells, are attracted to lung tissue and/or other organs by chemokine gradients and play an important role in the granuloma formation. T helper cells migrate from peripheral blood to the tissues due to expression of CXCR3 chemokine receptor on their surface. It interacts, e.g., with CXCL9/MIG, CXCL10/IP- 10, and CXCL11/I-TAC. Our study was aimed for determining the levels of CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC chemokines in peripheral blood of the patients with sarcoidosis, depending on the features of their clinical course before administration of immunosuppressive therapy. We studied peripheral blood plasma samples of the patients with sarcoidosis (n = 52). In 37% (19/52), they exhibited acute clinical manifestations, and 63% (33/52) had chronic sarcoidosis. The control group included peripheral blood samples from healthy volunteers (n = 22). The chemokine concentrations (pg/ml) were determined by multiplex analysis using xMAP technology (Luminex), and Milliplex MAP test system (Millipore, USA). In the patients with sarcoidosis, significantly higher levels of chemokines were shown relative to healthy volunteers: CXCL9, 4013.00 pg/ml vs 1142.00 pg/ml (p < 0.001); CXCL10, 565.90 pg/ml vs 196.60 pg/ml (p < 0.001); CXCL11, 230.20 pg/ml vs 121.10 pg/ml (p = 0.018). Plasma concentrations of CXCL9 and CXCL10 were significantly increased both in blood samples from patients with acute and chronic sarcoidosis compared to healthy volunteers, p < 0.001. The level of CXCL11 chemokine was significantly increased only in the patients with chronic sarcoidosis, compared to the healthy volunteers: respectively, 251.50 pg/ml and 121.10 pg/ml (p = 0.044). The levels of this chemokine correlated with the activity of angiotensin-converting enzyme (ACE), with r = 0.374; p = 0.042. The ACE level in sarcoidosis is considered a clinical and laboratory index of the disease activity. In acute sarcoidosis, the level of CXCL11 chemokine was not significantly higher than in healthy individuals, whereas the CXCL9 chemokine content was significantly increased and correlated with ACE activity (r = 0.762; p = 0.037). The level of CXCL9 chemokine was significantly decreased in patients with signs of fibrosis as compared with fibrosis-free patients (1839.88 pg/ml vs 4375.52 pg/ml, p = 0.035). Significantly higher levels of CXCL9 were detected in cases of systemic sarcoidosis, i.e. 6036.84 pg/ml, as compared with 1927.44 pg/ml in the patients without these signs (p = 0.018). Evaluation of clinical and laboratory diagnostic characteristics for plasma chemokine levels in sarcoidosis patients allowed to assess their sensitivity and specificity. The respective values were as follows: in acute sarcoidosis: for CXCL9, 84% and 95%; for CXCL10, 84% and 95%; for CXCL11, 74% and 59%. In chronic sarcoidosis, the respective values for CXCL9 were 82% and 72%; for CXCL10, 91% and 77%; for CXCL11, 79% and 55%, respectively. Thus, the determination of plasma CXCL9, CXCL10, and CXCL11 chemokines in sarcoidosis allows of understanding their role in development of the disease, e.g., recruitment of T helper cells from peripheral blood to the lung tissue, and granuloma formation. Clinical and immunological comparisons of CXCL9 levels in the peripheral blood of patients and characteristics of the clinical course of sarcoidosis indicate to the role of this diagnostic parameter for assessing the disease activity, signs of lung fibrosis, and systemic manifestations in this disease

CHEMOKINES CCL17 AND CCL22 IN SARCOIDOSIS

Various immune cells as well as related cytokines are involved in immunopathogenesis of sarcoidosis and mechanisms of granuloma development. Currently, a role for chemokines in sarcoidosis has been extensively investigated, which is paralleled with a search for key molecules necessary for recruiting immune cells to intrusion site and granuloma formation as well as affecting outcome of the latter. Our study was aimed for determining level of plasma CCL17/TARC and CCL22/MDC chemokines in patients with sarcoidosis who received no immunosuppressive therapy is of high priority for clarifying some aspects in underlying immunopathogenesis as well as seeking out for secure clinical and laboratory criteria for assessing activity and disease prognosis. We studied peripheral blood plasma samples of the patients with sarcoidosis (n = 52). In 37% (19/52), they exhibited acute clinical manifestations, and 63% (33/52) had chronic sarcoidosis. The control group included peripheral blood samples from healthy volunteers (n = 22). The chemokine concentrations (pg/ml) were determined by multiplex analysis using xMAP technology (Luminex), and Milliplex MAP test system (Millipore, USA). In the patients with sarcoidosis, significantly higher levels of chemokines were shown relative to healthy volunteers: CCL17 – 78.24 pg/ml vs 26.24 pg/ml, p < 0.001; CCL22 – 660.60 pg/ml vs 405.00 pg/ml, p < 0.001. Evaluation of clinical and laboratory diagnostic characteristics for plasma chemokine levels in sarcoidosis patients allowed to assess their sensitivity and specificity. The respective values were as follows: in acute sarcoidosis: for CCL17 – 63% and 78%, CCL22 – 63% and 91%; in chronic sarcoidosis: CCL17 – 58% and 83%, CCL22 – 67% and 86%, respectively. In chronic sarcoidosis the levels of this chemokine correlated with the activity of angiotensin-converting enzyme (ACE), for CCL17 (r = 0.530; p = 0.003), for CCL22 (r = 0.446; p = 0.014). Patients with systemic lesions vs no systemic lesions (sarcoidosis of the respiratory system only) had significantly elevated CCL17 level: 102.82 pg/ml vs 32.72 pg/ml, p = 0.011. The concentration of chemokine CCL17 was significantly increased in patients with vs without signs of hepatomegaly: 130.73 pg/ml vs 51.60 pg/ml, p = 0.022. Levels of chemokines was significantly increased in patients with vs without ultrasound signs of splenomegaly comprising: for CCL17 – 249.18 pg/ml vs 46.87 pg/ml, p = 0.002; for CCL22 – 1271.40 pg/ml vs 660.63 pg/ml, p = 0.003. Thus, it should be noted that the peripheral blood plasma level of chemokines CCL17 and CCL22 may be used as additional prognostic markers in chronic sarcoidosis with varying scoring of clinical signs including with/without systemic disease manifestations

Plasma cytokines in patients with COVID-19 during acute phase of the disease and following complete recovery

COVID-19, an infection caused by the new coronavirus SARS-CoV-2, is associated with a number of pathophysiological mechanisms, mobilizing a wide spectrum of biomolecules, mainly, cytokines.The purpose of this study was to evaluate levels of multiple cytokines in blood plasma from the patients with COVID-19 during acute phase of the disease, and upon complete recovery. Samples of peripheral blood plasma of 56 patients with COVID-19, 69 convalescents and 10 healthy individuals were examined. Concentrations of 46 molecules, such as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNα2, IFNγ, TNFα, TNFβ/ Lymphotoxin-α (LTA), CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROα, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine, IL-1ra, IL-10, EGF, FGF-2/FGF-basic, Flt3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGF-AB/ BB, TGF-α, VEGF-A were measured via xMAP multiplexing technology. Significantly increased levels of 18 cytokines were found in blood plasma from COVID-19 patients during acute phase of the disease (as compared to control group), i.e., IL-6, IL-7, IL-15, IL-27, TNFα, TNFβ/Lymphotoxin-α (LTA), CCL2/MCP-1, CCL7/MCP-3, CXCL1/GROα, CXCL8/IL-8, CXCL10/IP-10, CXCL9/MIG, IL-1rа, IL-10, M-CSF, GM-CSF, VEGF-A. We found a significant decrease of nearly all the mentioned cytokines in recovered patients, in comparison with those who had moderate, severe/extremely severe disease. Moreover, we revealed a significantly decreased level of 8 cytokines in plasma from convalescents, as compared with control group, i.e., IL-1α, IL-2, IL-9, IL-12 p40, IL-18, CCL22/MDC, Flt3 Ligand, TGF-α. Immune response caused by SARS-CoV-2 infection involves multiple cytokines, mostly, with pro-inflammatory effects. We have shown for the first time that the convalescence phase is characterized by significantly lower levels of cytokines which regulate cellular differentiation and hematopoiesis (in particular, lymphocytes, T-cells and NK-cells). Over acute phase of the disease, the levels of these cytokines did not change. We revealed a significant decrease of most plasma cytokines upon recovery as compared to acute phase. On the contrary, acute phase of the disease is accompanied by significant increase of both pro- and antiinflammatory cytokines in blood plasma

Sarcoidosis clinical picture governs alterations in type 17 T helper cell subset composition and cytokine profile

Immune cell hyperactivation along with cytokines they overproduce plays an important role in sarcoidosis and related disease pathogenesis. A central place in the immunopathogenesis of sarcoidosis is held by diverse cell-mediated reactions governed by T helper (Th) cell populations including Th17 subsets and relevant signature cytokines. We studied peripheral blood plasma samples of the patients with sarcoidosis (n = 123): 18% with acute and 82% with chronic course. The control group — samples from healthy volunteers (n = 43). T cell subset composition was assessed by flow cytometry. Cytokine concentrations (pg/mL) were measured by multiplex analysis using xMAP technology (Luminex). The level of “classical” Th17 turned out to be significantly reduced in acute vs chronic sarcoidosis: 28.3% vs 33.3% (p = 0.046). The level of “double-positive” Th17 (DP Th17) was significantly increased in chronic and acute vs control group: 31.7% and 34.2% vs 26.2% (p < 0.001 in both cases), without differences patient inter-group; “non-classical” Th17.1 were shown to have significantly reduced level only in chronic vs healthy subjects: 27.9% and 35.9% (p < 0.001). Clinical and laboratory diagnostic characteristics for blood DP Th17 levels in CD45RA-negative Th effector memory cells in sarcoidosis: in acute sarcoidosis vs healthy subjects, they were characterized by sensitivity — 82%; specificity — 71%, whereas in chronic: 67% and 56%, respectively. In patients with sarcoidosis vs healthy subjects were found to have significantly increased level of IL-12 (p70) — 1.3 vs 0.56, p = 0.028; IL-17A — 1.5 vs 0.43, p < 0.001; IFNγ — 4.1 vs 1.1, p < 0.001; TNFα — 21.7 vs 6.7, p < 0.001. Thus, CCR6+ Th17 and DP Th17 subsets and relevant signature cytokines are important in diagnostics of sarcoidosis of varying clinical course: a direct correlation was shown between the level of angiotensin-converting enzyme activity and percentage of memory DP Th17; disease progression vs regression had significantly reduced absolute number of total CD45RA- memory and CM Th17; extrapulmonary manifestations had a significantly increased percentage of DP Th17 CD45RA- and EM DP Th17; in chronic sarcoidosis are significantly increased concentration of IL-17A, IFNγ, IL-12 and positively correlation between IFNγ and the activity of angiotensin-converting enzyme

Changes in Anti-SARS-CoV-2 IgG Subclasses over Time and in Association with Disease Severity

IgG is the most prominent marker of post-COVID-19 immunity. Not only does this subtype mark the late stages of infection, but it also stays in the body for a timespan of at least 6 months. However, different IgG subclasses have different properties, and their roles in specific anti-COVID-19 responses have yet to be determined. We assessed the concentrations of IgG1, IgG2, IgG3, and IgG4 against different SARS-CoV-2 antigens (N protein, S protein RBD) using a specifically designed method and samples from 348 COVID-19 patients. We noted a statistically significant association between severity of COVID-19 infection and IgG concentrations (both total and subclasses). When assessing anti-N protein and anti-RBD IgG subclasses, we noted the importance of IgG3 as a subclass. Since it is often associated with early antiviral response, we presumed that the IgG3 subclass is the first high-affinity IgG antibody to be produced during COVID-19 infection