Search Results (498)

Cells of higher organisms express numerous cell surface proteins, and their spectrum and level of expression are directly related to cells’ functions. The technology of mass cell selection based on the surface protein expression levels may be highly important both for basic research and cell therapy applications. We have previously developed a method of magnetic selection of cells differing in surface marker expression levels, which we term here MACS-MEL (Magnetic-Assisted Cell Selection by Marker Expression Levels). The method demonstrated its effectiveness in the artificial model system, namely retrovirally transduced NIH 3T3 cells. However, whether it was also applicable to complex natural cell populations remained unclear. In the current study, we validated the MACS-MEL approach by separating mouse bone marrow (BM) cells into fractions according to the expression of pan-hematopoietic marker CD45. In the basic protocol, two-stage fractionation of CD45⁺ cells from BM was performed using selection of cells consecutively with 2 μL and 8 μL of anti-CD45 magnetic beads, resulting in isolation of CD45^high and CD45^int cell populations. To explore in full the potential of the method, the extended protocol was also tested, where a third selection stage with 30 μL of anti-CD45 beads was added. The isolated cell fractions were analyzed by flow cytometry for CD45 expression, as well for CD11b, Gr-1, CD117, CD115 and CD19 markers, while their in vitro progenitor function was assessed by quantitating colony-forming units (CFUs) in methyl cellulose. The results of analysis demonstrate that the isolated cell fractions significantly differed both in their surface phenotypes and CFU potential. In particular, cell fractions with progressively reduced CD45 expression were characterized by decreasing expression of myeloid differentiation markers CD11b and Gr-1, as well as B-lymphoid marker CD19. The expression of stem/progenitor cell marker CD117, on the contrary, significantly increased. The CFU frequency also strongly correlated with decrease in CD45 expression, while the differentiation potential of CFUs differed substantially in various cell fractions. In general, our results demonstrate that less differentiated hematopoietic cells in mouse BM studied using in vitro tests are characterized by lower CD45 expression levels, in full accordance with data obtained in human system. Successful validation of the MACS-MEL in a BM system, characterized by existence of multiple cell types and high phenotypic and functional heterogeneity, demonstrated the effectiveness, simplicity and affordability of this method. The MACS-MEL approach can be applied for mass selection of cells based on differential marker expression and may yield cell subsets suitable for advanced cell therapy applications. Full article

Pediatric acute myeloid leukemia (AML) is a highly heterogeneous malignancy, with cytogenetic and molecular abnormalities playing a critical role in determining prognosis and guiding treatment decisions. Despite therapeutic advances, patients with high-risk genetic mutations and translocations continue to experience suboptimal outcomes. As new targeted therapies emerge, the treatment of pediatric AML could undergo a paradigm shift, where “one-size-fits-all” chemotherapy is no longer the only frontline approach. Identifying genetic markers inform risk stratification and have greater impact on shaping the therapeutic approach, including the integration of targeted therapies such as FLT3 and menin inhibitors into frontline therapy. Furthermore, pediatric AML treatment options are being driven by recent discoveries in adult AML, broadening their clinical trials to include pediatric patients, in part due to the RACE for Children Act that went into effect in August 2020. This review identifies the most prevalent high-risk cytogenetic lesions in pediatric AML, emphasizing their incidence, prognostic significance, and implications for clinical management. By synthesizing current research on these key genetic abnormalities and their associated therapies, we aim to provide an updated perspective on the evolving landscape of high-risk pediatric AML management that can then lead to the establishment of an agile framework to rapidly evaluate, approve, and deploy novel agents. Full article

Accumulation of senescent endothelial cells (ECs) accelerates atherosclerosis, yet the molecular orchestrators linking metabolic dysregulation to epigenetic reprogramming during EC senescence remain elusive. Here, we investigated the specific role of Myeloid cell leukemia 1 (MCL1) in endothelial senescence and atherogenesis. Through integrative bioinformatics, MCL1 was identified as a candidate senescence regulator. MCL1 expression was validated in human carotid atherosclerotic plaques, ApoE^−/− mice, and multiple EC senescence models, primarily HRAS^G12V-induced senescent human umbilical vein endothelial cells (HUVECs). Loss- and gain-of-function assays were performed in HUVECs. Underlying metabolic and epigenetic mechanisms were explored using Seahorse extracellular flux analysis, lactate measurements, Co-IP and CUT&Tag sequencing. In vivo therapeutic potential was evaluated via AAV-mediated MCL1 knockdown in high-fat diet-fed ApoE^−/− mice. MCL1 expression was significantly upregulated in atherosclerotic plaques and senescent ECs. Functionally, MCL1 knockdown attenuated senescence markers (SA-β-gal, P21), suppressed the senescence-associated secretory phenotype (SASP), and restored cell proliferation, whereas MCL1 overexpression exacerbated EC senescence. Mechanistically, MCL1 promoted glycolytic reprogramming and intracellular lactate accumulation. This metabolic byproduct served as an epigenetic substrate for histone H4K12 lactylation (H4K12la), which specifically enriched at the CDKN1A (P21) promoter, correlating with its transcription. In vivo, AAV-mediated MCL1 silencing effectively reduced vascular H4K12la levels, alleviated vascular senescence, and substantially constrained atherosclerotic lesion areas. Our findings identify MCL1 as a pivotal regulator of endothelial senescence and atherosclerosis, operating through a metabolic-\–epigenetic mechanism involving glycolytic reprogramming, lactate accumulation, and H4K12la-associated P21 upregulation. These findings suggest that targeting the MCL1-associated pathway may represent a potential therapeutic strategy for atherosclerosis. Full article

The diagnosis and clinical management of myelodysplastic neoplasms are increasingly challenged by the disease’s inherent heterogeneity, particularly with respect to the diagnosis of low-grade variants. While standardized flow cytometric protocols traditionally rely on static biomarkers for lineage assignment, these often fail to capture the dynamic biological behavior of the malignant clone. This review synthesizes studies on the integration of functional biomarkers, specifically the nuclear proliferation marker Ki-67 and the anti-apoptotic protein Bcl-2, into the diagnostic and prognostic workflow. By utilizing high-dimensional multiparameter flow cytometry (MFC) and software-based maturation continuum analysis, the survival and growth kinetics of the myeloid, erythroid, and monocytic lineages can be quantified. These findings redefine myelodysplastic syndromes (MDS) as characterized by a significant decrease in cell-cycle progression and an increase in anti-apoptotic activity during early stages of maturation. Recent studies demonstrate that integrating the erythroid Ki-67 proliferation index as a fifth parameter into the conventional Ogata score dramatically improves diagnostic sensitivity for detecting MDS from 66% to 90% while maintaining 100% specificity. In particular, the sensitivity for detecting low-grade MDS improved from 56% to 91%. Additionally, a reduced erythroid Ki-67 index (≤28%) is a powerful independent predictor of transfusion dependence within 1 year. Beyond diagnostics, the introduction of the Bcl-2:Ki-67 ratio provides a superior metric for biological aggressiveness and a potential predictive tool for precision medicine. A high ratio identifies a quiescent, apoptosis-resistant cell population that is likely refractory to standard chemotherapy but is an ideal candidate for targeted Bcl-2 inhibition with Venetoclax. The integration of functional biomarkers bridges the gap between complex mutational landscapes and clinical manifestations. While digital imaging and artificial intelligence (AI) are beginning to automate blast enumeration and maturation analysis, functional kinetics may provide a necessary biological readout for personalized therapy. Full article

Oxidative stress is increasingly recognized as a key contributor to the biology of hematological malignancies. Excessive production of reactive oxygen species disrupts redox homeostasis, promotes genomic instability, alters cellular signaling pathways, and influences disease progression, therapeutic response, and resistance mechanisms. Among the downstream consequences of oxidative stress, lipid peroxidation represents a major source of cellular injury, generating reactive aldehydes capable of amplifying molecular damage. Malondialdehyde, a stable end product of lipid peroxidation, has emerged as one of the most widely investigated biomarkers of oxidative damage in hematologic cancers. This review summarizes current evidence regarding the role of malondialdehyde across major hematological malignancies, including acute and chronic myeloid leukemias, Philadelphia-negative myeloproliferative neoplasms, lymphomas, and multiple myeloma. Available evidence consistently demonstrates significantly elevated MDA levels across major hematological malignancies, including acute and chronic myeloid leukemias, Philadelphia-negative myeloproliferative neoplasms, lymphomas, and multiple myeloma. Increased MDA concentrations are frequently associated with disease activity, relapse, impaired antioxidant defenses, thrombotic complications, treatment resistance, and therapy-related toxicity. Furthermore, experimental studies indicate that MDA accumulation closely parallels ferroptosis induction and may serve as a pharmacodynamic marker of lipid peroxide-mediated cell death. Overall, the reviewed literature identifies MDA as one of the most consistent biomarkers of oxidative stress and lipid peroxidation in hematological cancers. Although methodological standardization remains necessary, MDA appears to have potential diagnostic, prognostic, and therapeutic relevance and may contribute to identifying redox vulnerabilities that can be exploited by emerging ferroptosis-based treatment strategies. Full article

Acute myeloid leukemia (AML) persistence is sustained by leukemic stem cells (LSCs) that survive metabolic deprivation, oxidative stress, hypoxia, proteotoxic burden, and therapeutic pressure. The integrated stress response (ISR) has emerged as a central adaptive network in this process. Through phosphorylation of a subunit of eukaryotic initiation factor 2 (eIF2α) and selective translation of activating transcription factor 4 (ATF4), the ISR coordinates stress-responsive transcriptional programs that may either preserve cellular fitness or promote apoptotic commitment, depending on the intensity, duration, and biological context of activation. In AML, ATF4 occupies a critical position at the interface between stemness, metabolic adaptation, redox control, ferroptosis resistance, and treatment response. In primitive leukemic compartments, ISR–ATF4 signaling appears to support stress tolerance, amino acid metabolism, serine biosynthesis, autophagy, and leukemic persistence. At the same time, pharmacologic or sustained ISR activation may lower the apoptotic threshold by inducing pro-apoptotic mediators such as CHOP, PUMA, and NOXA, thereby modulating MCL-1 dependency and enhancing sensitivity to venetoclax-based strategies. Conversely, adaptive ISR signaling may promote resistance through mechanisms such as ATP-binding cassette subfamily B member 1 (ABCB1) enhancer activation and mitochondrial stress tolerance. This duality creates a therapeutic paradox: ISR–ATF4 signaling may need to be inhibited in adaptive, resistance-promoting states but amplified in apoptosis-permissive contexts. This review discusses the biological and therapeutic relevance of ISR–ATF4 dysregulation in AML and highlights the need for biomarkers capable of distinguishing adaptive ATF4 dependency from inducible apoptotic vulnerability. Full article

Background: One of the key hallmarks of aging is the age-related decline in immune system function, accompanied by a chronic low-grade inflammation, or “inflammaging”. Simultaneously, a reduced capacity of immune cells to recognize and eliminate pathogens, along with immune exhaustion, is also defined as a sign of aging. Cynomolgus macaques (Macaca fascicularis) belong to a group of non-human primates evolutionarily close to humans and are often used for preclinical research. Methods: In this study, we performed mRNA sequencing of bone marrow and peripheral blood samples from young (5 years old) and old (over 19–21 years old) cynomolgus macaques to identify key markers of immunosenescence. Results: Although an increase in p16 expression was detected, we did not observe the increase in the senescence-associated secretory phenotype (SASP) cytokines reported in previous studies. Instead, we observed a transcriptional profile characterized by increased lymphocyte cytotoxic activity combined with a decrease in proinflammatory signaling, reduced markers of myeloid cells, and lowered sensitivity to pathogen-associated patterns. Similar changes were detected in both blood and bone marrow: decreased expression of naive T-cell markers (CCR7, LEF1, SELL, and FOXO1), reduced markers of the myeloid lineage—neutrophils and monocytes (CD177, CD14, CD163, FPR1, FPR2, and CXCR1)—and downregulation of genes belonging to different pattern-recognition receptor families (TLR1, TLR2, TLR4, TLR5, TLR6, TLR8, TLR10, IFIH1, CLEC4E, NOD2, NLRC4, NLRP12, NLRX1, and NAIP). In contrast, the group of old animals showed increased expression of markers associated with terminally differentiated cytotoxic lymphocytes (CD8+ T cells and NK cells): GZMB, PRF1, KLRK1, FASLG, TBX21, CCR5, and GNLY. Conclusions: Our findings offer new perspectives on the molecular mechanisms of age-associated immune dysregulation in non-human primates, serving as a baseline for selecting key candidate genes in subsequent functional investigations. Full article

Differentiating active ulcerative colitis (UC) from Crohn’s disease (CD) is one of the unmet needs addressed by biomarkers in inflammatory bowel disease (IBD). The immune landscapes of UC and CD differ, justifying the search for discriminatory markers and novel therapy targets among their mediators. Herein, 27 systemic cytokines were measured using flow cytometry-based methodology in 138 IBD patients, with an additional 21 being determined in 67 of the patients. Their discriminatory power was assessed individually and as exploratory multivariable signatures generated using logistic regression, hierarchical clustering, and principal component analysis. Eotaxin-1, macrophage inflammatory protein (MIP)-1β, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) showed fair discriminatory potential, while multivariable models performed better. Interleukin (IL)-1β, IL-4, and MIP-1α were strongly associated with CD, whereas IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), MIP-1β, and TRAIL were associated with UC. Active UC was characterized by mediators linked to eosinophil-, mastocyte-, and neutrophil-driven inflammation and tissue repair (eotaxin-1, IL-5, growth-regulated oncogene (GRO), MIP-1β, stem cell factor (SCF), GM-CSF, IL-1 receptor antagonist, TRAIL, stem cell growth factor (SCGF)-β, and cutaneous T cell-attracting chemokine (CTACK)), whereas active CD was associated with Th1/Th17 immunity, myeloid activation, fibrosis, angiogenesis, and neuroimmune remodeling (IL-1β, IL-12p70, IL-15, ‘regulated on activation, normal T-cell expressed and secreted’ (RANTES), MIP-1α, stromal cell-derived factor (SDF)-1α, nerve growth factor β (β-NGF), and leukemia inhibitory factor (LIF)). In conclusion, integrated circulating immune signatures identify several understudied cytokines as potential contributors to disease-specific pathways and show potential in distinguishing active UC from CD warranting further mechanistic studies and independent validation in larger cohorts. Full article

Fms-related receptor tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations occur in approximately 20–25% of patients with acute myeloid leukemia (AML) and are associated with increased relapse risk and inferior survival outcomes. Although measurable residual disease (MRD) has become a key prognostic tool for guiding post-remission treatment decisions, FLT3-ITD was historically considered a suboptimal MRD marker because of its subclonal nature, structural heterogeneity and dynamic behavior during disease evolution. In addition, FLT3-ITD has not yet been fully integrated into routine MRD monitoring due to methodological limitations and a lack of standardized workflows. The latest European LeukemiaNet (ELN)-DAVID 2025 recommendations stressed the use of ultra-high sensitivity (UHS) next-generation sequencing (NGS) technologies to detect FLT3-ITD MRD with improved precision, enabling reliable longitudinal tracking of patient-specific clones at very low variant allele frequencies (VAF). Indeed, despite prospective evidence supporting this approach remaining limited, FLT3-ITD-based MRD monitoring is emerging as a clinically relevant prognostic indicator, contributing to the identification of patients at increased risk of relapse and refining risk stratification, while also informing therapeutic decision-making, particularly in the peri-transplant setting. The present review summarizes the biological underpinnings of FLT3-ITD mutated (FLT3-ITD^mut) AML, discusses the methodological challenges of MRD detection, and critically evaluates the evolving role of MRD in refining relapse prediction, supporting post-remission therapy tailoring, and contributing to a harmonized framework for FLT3-ITD^mut AML management. Full article

Background/Objectives: Flow-cytometric readouts in acute myeloid leukemia (AML) may depend on specimen source and processing. We assessed whether sample source (peripheral blood [PB] vs. bone marrow [BM]) and cryopreservation influence immune-evasion-related markers on AML blasts. Methods: Paired PB and BM samples from 19 adults were analyzed fresh and after cryopreservation. Viable CD45^dim singlets were assessed for ULBP1-6, MICA/B, CD34, CD44, CD70, CD80, and CD184 using median fluorescence intensity (MFI) and relative fluorescence index (RFI). Paired comparisons used two-sided Wilcoxon signed-rank tests with Benjamini–Hochberg correction across six markers within each endpoint and comparison. Discordance was also assessed using a predefined conventional 20% reference threshold. Results: After adjustment, RFI did not differ between fresh and cryopreserved samples within PB or BM, or between fresh PB and BM. In cryopreserved samples, PB and BM differed for CD70 (q = 0.042) and ULBP1-6 (q = 0.028). Cryopreservation increased MFI for CD184 and ULBP1-6 in PB and BM and for MICA/B in PB; no PB-BM MFI difference remained significant. Threshold-based classification was discordant for CD34, CD44, CD80, CD184, MICA/B, and ULBP1-6 in 5.3–38.9% of pairs, whereas CD70 showed none. Conclusions: Cohort-level differences were limited to specific markers and conditions, while patient-level threshold discordance persisted. Sample source and cryopreservation should therefore be considered when interpreting threshold-based classifications. Full article

Background/Objectives: Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease (IBD) lacking therapies that directly promote mucosal healing, an important clinical endpoint and therapeutic goal for achieving remission and improved long-term outcomes. Epithelial restitution is a key component of effective mucosal healing. EPICERTIN, a novel biotherapeutic candidate, has previously demonstrated epithelial repair activity in dextran sulfate sodium (DSS)-induced colitis models and enhanced epithelial cell viability in human IBD colon explants. To further substantiate its therapeutic efficacy in a UC-relevant context, we evaluated EPICERTIN in acute oxazolone (OXA)-induced colitis in BALB/c mice, a model reflecting the adaptive immune-driven inflammation and histopathologic characteristics of human UC. Methods: Orally administered EPICERTIN, at escalating doses (0.3 µg, 3 µg, and 30 µg), was assessed for therapeutic efficacy in male and female mice through body weight, Disease Activity Index (DAI) scores, and histopathology. Wound healing and immune impacts were examined using qRT-PCR, Imaging Mass Cytometry (IMC), and Cytometry by Time of Flight (CyTOF) in male mice. Results: EPICERTIN effectively mitigated acute OXA-induced colitis. The 3 µg dose provided the greatest benefit, improving body weight recovery and reducing DAI and histopathological damage scores. Treatment reduced Il1b while increasing Cdh1 expression, accompanied by higher levels of epithelial markers (pan-cytokeratin (PanCK), E-cadherin, epithelial cell adhesion marker (EpCAM)) and decreased fibrotic markers (collagen type I, alpha-smooth muscle actin (α-SMA), fibronectin). Moreover, EPICERTIN reduced inflammatory lymphoid and myeloid cells while increasing γδ T cells in the colon lamina propria. Conclusions: Collectively, these results demonstrate that EPICERTIN promotes epithelial restitution, suppresses inflammation, and supports mucosal healing, substantiating its therapeutic potential for UC. Full article

Mass cytometers can record 40–50 parameters per single cell for millions of cells in a sample. Many methods have been developed to cluster phenotypically similar cells within cytometry data, but there are fewer methods to visualize activity and interactions of pairs of proteins across these populations. We have developed a workflow for analyzing correlations associated with predefined populations. By clustering blood samples from acute myeloid leukemia (AML) patients and normal controls using an established algorithm, we obtained a minimum spanning tree of clusters of single cells. Using surface marker expression, we identified clusters on the tree that belonged to phenotypes of interest. Next, we computed correlations between pairs of proteins in each cluster. We developed a novel, coherent, probability-based statistic to test differences between vectors of correlation coefficients. By comparing all combinations of the normal controls under the statistic, we created an empirical distribution that could provide a conservative threshold of differential correlation. Using this empirically derived distribution to define significance, we compared pooled samples from AML subtypes and normal controls to detect differential correlations. Given the structure present within this cytometry dataset, we found it natural to consider correlations in this manner versus aggregating all data and computing a single correlation. Our approach has the advantage that we can localize the statistical measure to determine contributions from particular phenotypic populations. Differentially correlated pairs of proteins can be further explored as possible testable hypotheses by considering a population’s distribution of correlation coefficients or biaxially plotting protein expressions within individual cells in a given population. Full article

Background/Objectives: Brain arteriovenous malformations (bAVMs) are complex cerebrovascular lesions that carry a substantial risk of intracranial hemorrhage, yet the biological mechanisms underlying vascular remodeling remain incompletely understood. As increasing evidence suggests that inflammatory processes contribute to vascular remodeling and bAVM progression, this study aimed to characterize the cellular composition of the immune microenvironment in human bAVMs at single-cell resolution and to identify immune cell populations associated with transcriptional programs related to angiogenesis and extracellular matrix remodeling. Methods: Publicly accessible single-cell RNA sequencing data from five human bAVM samples and five control brain specimens were analyzed. After quality control and data integration, immune cell types were annotated based on canonical marker gene expression. To assess biological processes relevant to vascular remodeling, the expression of predefined gene sets associated with angiogenesis and extracellular matrix remodeling, derived from the Molecular Signatures Database (MSigDB), was quantified across cell populations using rank-based gene set scoring (UCell). Differences between bAVM and control samples were evaluated at the sample level using the Wilcoxon rank-sum test. For each immune cell type, mean UCell scores were calculated per biological sample and compared between groups. To account for multiple comparisons across cell types, p-values were adjusted using the Benjamini–Hochberg procedure. Results: After quality control, 46,360 immune cells were analyzed. Compared with control tissue, bAVMs showed numerically higher proportions of lymphoid (activated T cells and CD8 T cells) and myeloid populations (M1-like macrophages, monocytes, and dendritic cells). Angiogenesis-related transcriptional activity was highest in myeloid cells and significantly increased in bAVMs, particularly in M1-like macrophages (UCell score 0.22 vs. 0.13), dendritic cells (0.16 vs. 0.10) and monocytes (0.20 vs. 0.15), whereas proliferating CD8 T cells showed a lower score (0.04 vs. 0.05; adjusted p = 0.040 for all four). Extracellular matrix-related programs showed a similar but weaker and non-significant pattern in myeloid populations (e.g., monocytes, dendritic cells, microglia-like cells; adjusted p = 0.09). Conclusions: Our findings identify myeloid cells, particularly M1-like macrophages, monocytes and dendritic cells, as important immune populations associated with angiogenesis in bAVMs. These findings highlight a potential role of immune-driven vascular remodeling in the pathophysiology of bAVMs. Full article

Soft tissue sarcomas are rare, biologically diverse mesenchymal malignancies in which pharmacologic resistance cannot be explained by a single unifying mechanism. In this narrative review, resistance is conceptualized as a dynamic, multilayered process shaped by histologic subtype, genomic architecture, transcriptional plasticity, the tumor microenvironment, and treatment-driven selective pressure. Resistance to conventional chemotherapy arises through both intrinsic and acquired mechanisms, including altered drug transport and metabolism, enhanced DNA damage responses, impaired apoptotic signaling, clonal selection, and the emergence of therapy-persistent cellular states. By contrast, resistance to targeted and epigenetic therapies more often reflects adaptive bypass signaling, lineage reprogramming, and incomplete identification of subtype-specific dependencies than secondary on-target alterations alone. The tumor microenvironment further contributes to therapeutic failure through hypoxia, extracellular matrix-mediated barriers, abnormal vascularization, myeloid-dominant immunosuppression, and immune exclusion, thereby helping explain the modest and histology-dependent activity of immune checkpoint inhibitors in soft tissue sarcoma. This review also differentiates baseline predictive biomarkers from dynamic resistance-monitoring tools, underscoring the potential—despite still limited clinical maturity—of pharmacogenomic markers, immune signatures, tertiary lymphoid structures, circulating tumor DNA, and circulating methylation-based approaches. Finally, emerging strategies to overcome resistance are examined, including mechanism-based combinations, biomarker-guided treatment selection, synthetic lethality, functional precision platforms, and adaptive histology-specific trial designs. Collectively, these observations support a view of resistance in soft tissue sarcoma as a context-dependent biological process that demands integrated, subtype-aware, and translationally grounded therapeutic strategies. Full article

The COVID-19 pandemic underscored the need to better characterize immune and molecular responses following SARS-CoV-2 infection and vaccination. Beyond antibody and cellular immunity, COVID-19 involves oxidative stress and DNA damage, affecting repair mechanisms and metabolic adaptation linked to immune resilience. Here, we present a multidimensional analysis of 20 individuals who recovered from mild COVID-19, integrating clinical features with humoral and cellular immune responses, T cell and myeloid phenotypes, oxidative stress, DNA damage, and metabolomic and lipidomic profiles. Although most individual parameters fell within physiological ranges, network modeling revealed structured associations spanning multiple biological domains. A central finding was a coherent cluster organized around vaccine dose number, linking anti-Spike antibody titers, oxidative stress, bioenergetic signatures, and granulocyte activation. Higher vaccination was associated with stronger humoral responses, lower oxidative stress, and a more balanced myeloid–metabolic profile, suggesting a potential protective role extending beyond antibody induction. Additional associations linked symptom patterns to T cell differentiation states, anti-nucleocapsid responses to systemic inflammation, and anaerobic signatures to DNA damage markers, revealing interconnections between immunometabolism, clinical expression, and genomic stress. Despite the small sample size, these findings offer a preliminary systems-level perspective on mild COVID-19 recovery and illustrate the value of integrative exploratory frameworks in infectious disease research, laying the groundwork for validation in larger longitudinal cohorts. Full article