Alexandra Baldzhieva

The immune response to SARS-CoV-2 infection involves significant alterations in the phenotype and function of natural killer (NK) cells. This study aimed to investigate the dynamic changes in NK cell subsets during COVID-19 by analyzing their activation and inhibitory markers [CD3, CD14, CD16, CD19, CD25, CD45, CD56, CD57, CD69, CD159a (NKG2A), CD159c (NKG2C), CD314 (NKG2D), CD335 (NKp46)], cytotoxic potential (perforin, interferon-gamma, granzyme B), and direct cytotoxicity against a newly genetically modified K562 cell line. Peripheral blood samples were collected from COVID-19 patients on days 3–5 and day 30 post-symptom onset and were compared to healthy controls. 16-color flow cytometry analysis revealed distinct shifts in NK cell subpopulations, characterized by increased expression of the inhibitory receptor NKG2A and the activating receptors NKG2D and NKG2C, particularly in the CD56⁺CD16⁻ subset. Elevated IFN-γ production on day 30 suggested a recovery-phase immune response, while the persistent upregulation of NKG2A indicated an ongoing regulatory mechanism. The CD16⁺CD56⁻ subpopulation exhibited increased expression of the markers CD69 and CD25 over time; however, its cytotoxic potential, assessed through granzyme B levels and direct cytotoxicity assays, remained lower than that of healthy controls. Significant correlations were observed between CD57 and CD69 expression, as well as NKp46 and IFN-γ production, highlighting a coordinated balance between activation and regulatory mechanisms. These findings suggest that NK cells undergo functional adaptation during COVID-19, displaying signs of partial exhaustion while retaining antiviral potential. Understanding the interplay between NK cell activation and suppression may provide valuable insights into immune dysregulation in COVID-19 and inform potential therapeutic interventions.

Measurable Residual Disease (MRD) assessment in pediatric acute lymphoblastic leukemia (ALL) is crucial for relapse prediction and treatment guidance. Multiparameter flow cytometry (MFC) enhances detection but faces limitations due to insufficient leukemia-associated immunophenotypes (LAIPs) and antigen modulation. This study explores new markers to improve MFC-based MRD detection in B-cell precursor ALL (BCP-ALL). Expression-patterns of seven aberrancy markers, i.e., CD44, CD304, CD73, CD86, CD123, CD99, CD58, and one B-cell maturation marker, CD22, were studied in 143 samples with leukemic-blasts from sixty-one childhood BCP-ALL patients and in hematogones of 20 non-leukemic bone marrow (BM) samples using fourteen-color MFC. The highest relative frequences of LAIPs amounted to 82.50%, reported for CD99 and CD58, followed by CD44 (81.10%), CD73 (76.20%), CD22 (73.40%), CD304 and CD86 (68.50%), while the lowest relative frequence was CD123 (44.40%). Differential expression of CD58, CD304, and CD73 in diagnostic samples was highly significant (p < 0.01) between pre-B-I, pre-B-II, immature B cells, and BCP-ALL blasts. In MRD-positive samples CD73 showed significantly high (p < 0.01) differential expression between all stages of hematogones and residual blasts, followed by CD304, CD58, and CD22. CD73 and CD304 were identified as the most reliable among the tested markers for distinguishing both diagnostic and MRD blasts from normal B cell precursors.

Alginates are anionic polysaccharides present in the cell walls of brown seaweeds. Various biological activities of alginate and its derivatives have been described. In this study, we assessed the potential of alginate obtained from Ericaria crinita (formerly Cystoseira crinita) to scavenge free radicals and function as a ferric ion reductor. The anti-inflammatory effect on the serum levels of TNF-α, IL-1β, IL-6, and IL-10 of rats with LPS-induced systemic inflammation after 14 days of treatment was also examined. Ericaria crinita alginate showed antioxidant activities of IC₅₀ = 505 µg/mL (DPPH) and OD₇₀₀ > 2 (ferric reducing power). A significant decrease in serum levels of IL-1β was observed only in animals treated with the polysaccharide at a dose of 100 mg/kg bw. Both doses of E. crinita alginate (25 and 100 mg/kg bw) significantly reduced the serum concentrations of pro-inflammatory cytokines TNF-α and IL-6, but no statistical significance was observed in the levels of the anti-inflammatory cytokine IL-10. Our findings show the potential of E. crinita alginate to act as an antioxidant and anti-inflammatory agent. It is likely that the exhibited antioxidant ability of the polysaccharide contributes to its antiphlogistic effects. More in-depth studies are needed to fully understand the specific mechanisms and the molecular pathways involved in these activities.

Background: The SARS-CoV-2 virus as well as the COVID-19 mRNA vaccines cause an increased production of proinflammatory cytokines. Aim: We investigated the relationship between ACE2, CTSL, AngII, TNFα and the serum levels of IL-6, IL-10, IL-33, IL-28A, CD40L, total IgM, IgG, IgA and absolute count of T- and B-lymphocytes in COVID-19 patients, vaccinees and healthy individuals. Methods: We measured the serum levels ACE2, AngII, CTSL, TNFα and humoral biomarkers (CD40L, IL-28A, IL-10, IL-33) by the ELISA method. Immunophenotyping of lymphocyte subpopulations was performed by flow cytometry. Total serum immunoglobulins were analyzed by the turbidimetry method. Results: The results established an increase in the total serum levels for ACE2, CTSL, AngII and TNFα by severely ill patients and vaccinated persons. The correlation analysis described a positive relationship between ACE2 and proinflammatory cytokines IL-33 (r = 0.539) and CD40L (r = 0.520), a positive relationship between AngII and CD40L (r = 0.504), as well as between AngII and IL-33 (r = 0.416), and a positive relationship between CTSL, total IgA (r = 0.437) and IL-28A (r = 0.592). Correlation analysis confirmed only two of the positive relationships between TNFα and IL-28A (r = 0.491) and CD40L (r = 0.458). Conclusions: In summary, the findings presented in this study unveil a complex web of interactions within the immune system in response to SARS-CoV-2 infection and vaccination.

Fucoidans are sulfated polysaccharides detected mainly in the cell walls of brown seaweeds. Here, we examined the effects of single doses of fucoidan derived from Ericaria crinita (formerly Cystoseira crinita) on carrageenan-induced paw inflammation in rats. The serum levels of TNF-α, IL-1β, IL-6, and IL-10 of rats with LPS-induced systemic inflammation after 14 days of treatment were also evaluated. Subchronic treatment with fucoidan from E. crinita attenuated the inflammation during the late phase of the degraded carrageenan-induced paw edema (3rd to 5th hour after carrageenan injection) with peak activity at the 3rd hour after the application. Both doses of fucoidan from E. crinita (25 and 50 mg/kg bw) significantly decreased the levels of all tested pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in the serum of rats with a model of system inflammation but had no effect on the anti-inflammatory cytokine IL-10. The results showed that the repeated application of fucoidan has a more prominent effect on the levels of some pro-inflammatory cytokines in serum in comparison to a single dose of the sulfated polysaccharide. This reveals the potential of E. crinita fucoidan as an anti-inflammatory agent. Furthermore, E. crinita fucoidan exhibited in vitro antioxidant capacity, determined by 2,2-diphenyl-1-picryl-hydrazyl radical scavenging and ferric reducing antioxidant power assays as follows: IC₅₀ = 412 µg/mL and 118.72 μM Trolox equivalent/g, respectively.

The first epidemiological wave of the incidence of COVID-19 in Bulgaria was registered in June 2020. After the wave peak, we conducted a study in persons diagnosed with COVID-19 (N = 52). They were assessed with the anxiety–depressive scale (ADS), including basic (BS), vegetative (VS), conversion (CS), obsessive–phobic (OPS), and depressive (DS) symptoms. ADS assessment of individuals diagnosed with SARS-CoV-2 indicated a correlation between OPS and IL-33 values. IL-10 levels were higher than reference ranges in all patients. Multiple linear regression analyses demonstrated that combination of CS and OPS explained 28% of IL-33 levels, while combination of symptoms from all ADS dimensions explained 24% of IL-33 levels. It was also found that 21% of IL-28A levels was explained from the combination by all ADS dimensions, whereas OPS was the predictor for lower concentrations. The obtained results revealed meaningful correlations between psycho neuro–immunological factors in pathogenesis of illness from the coronavirus infection.

Improvements in medical care have turned severe diseases into chronic conditions, but often their treatment and the use of medical devices are related to specific complications. Here, we present a clinical case of a long-term dialysis patient who was infected with a rare opportunistic infectious agent—Gordonia sputi. In recent years, the incidence of Gordonia spp. infections in immunocompromised patients with central venous catheters (CVC) has appeared to rise. The isolation and identification of Gordonia spp. are challenging and require modern techniques. In addition, the treatment is usually persistent and often results in CVC extraction, which is associated with further risk and costs for the patient. We also studied the alterations in the immune status of the patient caused by long-term renal replacement therapy and persistent hepatitis C virus infection. Antibiotic therapy and immunostimulation with Inosine pranobex lead to successful eradication of the infection without the need for CVC replacement.

Minimal residual disease refers to a leukemia cell population that is resistant to chemotherapy or radiotherapy and leads to disease relapse. The assessment of MRD is crucial for making an accurate prognosis of the disease and for the choice of optimal treatment strategy. Here, we review the advantages and disadvantages of the available genetic and phenotypic methods and focus on the multiparametric flow cytometry as a promising method with greater sensitivity, speed, and standardization options. In addition, we discuss how the application of automated data analysis outweighs the use of complex combinations of windows and gates in classical analysis, thus eliminating subjective evaluation.

Improvement in medical care has turned severe diseases into chronic conditions, but often their treatment and use of medical devices are related to specific complications. Here we present a clinical case of a long-term dialysis patient, who is infected with a rare opportunistic infectious agent – Gordonia sputi. In recent years the incidence of Gordonia spp. infections in immunocompromised patients with central venous catheters (CVC) appear to rise. Their isolation and identification are challenging and require modern techniques. In addition, the treatment is usually persistent and often results in CVC extraction, which is associated with further risk and costs for the patient. We also studied the alternations in the immune status of the patient caused by long-term renal replacement therapy and persistent HCV infection. Antibiotic therapy and immunostimulation with Inosine pranobex lead to successful eradication of the infection without the need for CVC replacement.

The aim of this study was to identify the chemical composition and sequential structure of alginate isolated from C. crinita harvested in the Bulgarian Black Sea, as well as its effects in histamine-induced paw inflammation in rats. The serum levels of TNF-α, IL-1β, IL-6, and IL-10 in rats with systemic inflammation, and the levels of TNF-α in a model of acute peritonitis in rats were also investigated. The structural characterization of the polysaccharide was obtained by FTIR, SEC-MALS, and ¹H NMR. The extracted alginate had an M/G ratio of 1.018, a molecular weight of 7.31 × 10⁴ g/mol, and a polydispersity index of 1.38. C. crinita alginate in doses of 25 and 100 mg/kg showed well-defined anti-inflammatory activity in the model of paw edema. A significant decrease in serum levels of IL-1β was observed only in animals treated with C. crinita alginate in a dose of 25 mg/kg bw. The concentrations of TNF-α and IL-6 in serum were significantly reduced in rats treated with both doses of the polysaccharide, but no statistical significance was observed in the levels of the anti-inflammatory cytokine IL-10. A single dose of alginate did not significantly alter the levels of the pro-inflammatory cytokine TNF-α in the peritoneal fluid of rats with a model of peritonitis.