ABSTRACT Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

ABSTRACT Background The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is controversial. The aim of this study was to investigate whether renal biopsies evaluated by routine immunofluorescence on frozen tissue (IF-Frozen) would yield adequate findings to confirm diagnoses when the IF-Paraffin technique was applied. Methods To show immunoglobulins, complement components, and light chains, 55 native renal biopsies were subjected to IF-Paraffin and IF-Frozen staining techniques. The intensity of the staining was compared, and the sensitivity and specificity were calculated. Results The IF-Paraffin technique showed a sensitivity of 89%, 81%, 86%, 30%, 71%, 60%, and 77% for IgG, IgM, IgA, C1q, C3, κ, and λ, respectively, whereas specificity was 91%, 100%, 100%, 96%, 94%, 98%, and 100%. It showed diagnostic findings in 87% of cases. Compared to cases that had both IF-Paraffin and IF-Frozen staining techniques, 43 of 55 showed either equal intensity for the diagnostic immunoglobulin/complement or a little difference. Conclusions Direct immunofluorescence on formalin-fixed, paraffin-embedded sections cannot replace immunofluorescence on frozen sections in the assessment of renal biopsies, but may be a “salvage technique” when frozen tissue is insufficient or unavailable and must be interpreted with great caution.

ABSTRACT Myeloperoxidase (MPO) is a pro-oxidant enzyme mainly found in the azurophilic granules of neutrophils. It not only displays a key role in the intracellular microbial killing process but also contributes to the extracellular clearance of several pathogens. This study aimed to detect MPO in cutaneous leukocytoclastic vasculitis (LCV) using immunohistochemistry. We retrospectively collected 22 confirmed cases of skin LCV diagnosed in our pathology department over 11 years (2012–2023). Immunohistochemistry was performed using anti-myeloperoxidase antibody (Leica clone 59A5) on the LeicaBond MAX automated system, following manufacturer’s instructions. Two pathologists assessed immunohistochemical staining, scoring intensity as weak (+), moderate (++), or strong (+++). Patients’ mean age was 56.9 years, with a male-to-female ratio of 1.18. Pathologically, vasculitis involved small blood vessels in all cases. Immunohistochemical analysis showed granular positive MPO staining in 59.1% of cases. Staining intensity was weak in 46.15%, moderate in 46.15%, and strong in 7.69%. Staining was patchy in 84.62% and diffuse in 15.38% of cases. MPO expression, detected in 59.1% of cutaneous LCV tissues, exhibited a patchy and peri-vascular distribution. It holds potential as a diagnostic marker for patients with early or minor histological changes.

ABSTRACT Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) susceptibility depends on a complex interaction between environmental and genetic factors. Genes for tumor necrosis factor alpha (TNF-α) and toll-like receptors (TLRs) have been incorporated into the pathophysiology of autoimmune disorders. Our aim is to assess the association between TLR7 (rs179009) and TNF-α (rs1800629) polymorphisms and susceptibility to autoimmune thyroid disorders. One-hundred ninety-nine individuals, divided into 68 HT patients in group I, 57 GD patients in group II, and 74 age- and gender-matched healthy subjects in group III, underwent laboratory investigations, including the detection of TLR7 and TNF-α polymorphisms using real-time PCR technique. TLR7 (rs179009) genotypes, A/G and G/G, were significantly more prevalent in HT patients (group I) compared to normal controls. Meanwhile, TNF-α (rs1800629) genotypes in GD patients (group II) showed a six fold increase in the risk of the disease in the G/A and A/A genotypes. Our findings propose the fact that the polymorphisms of TLR7 (rs179009) play a role in the susceptibility and the development of Hashimoto’s thyroiditis, whereas TNF-α (rs1800629) polymorphisms play a role in the susceptibility and development of Graves’ disease.

ABSTRACT Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the main treatment for metastatic renal cell carcinoma (mRCC). Development of resistance is a major obstacle against therapy success. The aim of this study was to assess annexin A2 and CD163+ tumor associated macrophages (TAMs) immunohistochemical expression in 50 mRCC cases as regard to patients’ prognosis and Sunitinib response. Also, to assess the correlation between annexin A2 and TAMs expression. High annexin A2 expression and TAMs density were associated with serum calcium level (P = 0.024 and 0.037, respectively), larger tumor size (P < 0.001), high tumor grade (P = 0.014 and <0.001, respectively), and the presence of tumor necrosis (P < 0.001). High annexin A2 and TAMs expressions were related to shorter patients’ overall survival (P = 0.009 and 0.001, respectively) and progression-free survival (P = 0.003 and 0.001, respectively). Annexin A2 was correlated with TAMs density (r = 0.890). Annexin A2 and TAMs are associated with poor prognostic parameters in mRCC patients, including high nuclear grade, increased tumor size, and the presence of tumor necrosis, together with shorter patients’ survivals and poor response to Sunitinib. Annexin A2 expression is correlated with TAMs density suggesting immunomodulatory role of annexin A2.

ABSTRACT In view of multiplicity of carcinogenic pathways of gastric carcinoma (GC), poor survival and chemotherapy resistance, more analysis of these pathways is required for prediction of prognosis and developing new therapeutic targets. Knocking down of RORα; induces tumor cell proliferation and epithelial–mesenchymal transition (EMT). LAPTM4B has been suggested to be associated with EMT which promote tumor invasion. This work aimed to investigate prognostic role of RORα, LAPTM4B, and E-Cadherin expression in GC. This retrospective immunohistochemical study assesses the expression of RORα, LAPTM4B, and E-Cadherin in 73 primary gastric carcinomas. Low RORα and high LAPTM4B expression in GC cases were associated with unfavorable prognostic factors such as positive lymph nodes, and high tumor budding. E-Cadherin heterogeneous staining was associated with poor prognostic criteria, such as diffuse type GC and high tumor budding. Low RORα, high LAPTM4B, and heterogeneous E-Cadherin were the most common immunohistochemical profile in GC cases. Low RORα expression showed poor prognostic impact on overall patient survival. In conclusion, RORα and LAPTM4B may have crucial role in GC aggressiveness. The predominance of low RORα, high LAPTM4B, and heterogeneous or negative E-Cadherin immunohistochemical profile in GC cases with unfavorable pathological parameters suggested that this profile may predict tumor behavior.

ABSTRACT Addictive disorders are associated with systemic and central nervous system inflammation, which may be important for the onset and development of these diseases. Although lymphocyte-related parameters have recently been studied in alcohol use disorder (AUD), systemic immune-inflammation index (SII) and systemic inflammatory response index (SIRI) haven’t. Lymphocyte-related ratios, SII and SIRI levels were evaluated between AUD and healthy controls (HC) in this study. It was a retrospective and cross-sectional study. This study included 72 patients with AUD and 184 individuals in the HC group. Lymphocyte related ratios such as neutrophil to lymphocyte ratio (NLR), monocyte to lymphocyte ratio (MLR) and platelet to lymphocyte ratio (PLR), SII and SIRI values were compared. Compared to HC group, NLR (p < 0.001), MLR (p < 0.001), and SIRI (p < 0.001) levels were significantly higher in AUD group. There was also a significant relationship between NLR and AST/ALT ratio in the AUD group (p = 0.022). The results of this study support that AUD is a chronic inflammatory psychiatric disorder. In addition, it may be useful to evaluate these markers in relation to liver enzymes in patients with AUD, as alcohol consumption causes liver damage. These markers may also be used in future studies to assess treatment response and disease severity.

ABSTRACT We aim to assess the clinical impact of circulating levels of sCD163, FoxP3, IGF-1 in LSCC patients (Laryngeal Squamous Cell Carcinoma). The concentrations of sCD163, FoxP3, and IGF-1 were measured using ELISA test in the serum samples collected from 70 pretreatment LSCC patients and 70 age and sex-matched healthy controls. Statistical analysis was performed using ANOVA to compare the two groups, and the correlation between markers and clinical parameters. Receiver-Operator Characteristic (ROC) curve analysis was conducted to determine the optimal cutoff values and evaluate the diagnostic impact of these markers. Significant differences in the levels of sCD163, FoxP3, and IGF-1 were observed between LSCC patients and the control group, with respective p-values of 0.01, 0.022, <0.0001. The determined cutoff values for sCD163, FoxP3, IGF-1 concentrations were 314.55 ng/mL, 1.69 ng/mL, and 1.69 ng/mL, respectively. The corresponding area under the curve (AUC) values were 0.67 (95% CI: 0.57–0.76), 0.70 (95% CI: 0.61–0.80), 0.84 (95% CI: 0.76–0.92), respectively. Furthermore, it was found that IGF-1 concentrations exceeding 125.20 ng/mL were positively correlated with lymph node metastasis. Elevated serum levels of sCD163, FoxP3 and IGF-1 are associated with the diagnosis of LSCC. IGF-1 appears to be the most promising indicator for the LSCC progression.

ABSTRACT Gastric carcinoma (GC) is one of the most prevalent cancers worldwide and the fourth leading cause of cancer-related death. Studying the molecular profile of GC is essential for developing targeted therapies. β-catenin, Tenascin, and Fascin expression are among the molecular abnormalities that are claimed to cause GC progression and chemoresistance. Therefore, they could be used as potential therapeutic targets. This study aimed to evaluate β-catenin, Tenascin, and Fascin expression and their possible roles as prognostic and predictive biomarkers in GC using immunohistochemistry. This retrospective study included 84 GC cases. Tissue microarrays were constructed, followed by β-catenin, Tenascin, and Fascin immunostaining. Their expression was assessed and compared with clinicopathological parameters and survival data. The study results revealed that β-catenin nucleocytoplasmic expression, positive Tenascin, and Fascin expressions were detected in 86.9%, 70%, and 59.5% of cases, respectively. Their expression was significantly associated with poor prognostic parameters, such as deeper tumor invasion, lymph node metastasis, advanced pathological stage, vascular invasion, positive omental nodules, poor response to chemotherapy, and short overall survival. Hence, nucleocytoplasmic β-catenin expression together with Tenascin and Fascin positivity can be potential prognostic and predictive markers, and they can be used as therapeutic targets for GC.