Negative Marker Research Articles

(PM-02) GENITOURINARY TUBERCULOSIS IN MALE PATIENTS: A CASE SERIES FROM A FEDERAL UNIVERSITY HOSPITAL

Abstract Introduction Tuberculosis is a significant global public health threat, causing 1.2 million deaths in 2019. In Brazil, the incidence of the disease in the same year was 46 new cases per 100,000 inhabitants. Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis, resulting from the spread of mycobacteria through hematogenous dissemination from a latent chronic pulmonary infection or by local spread. Objective This study aims to present a case series of genitourinary tuberculosis in male patients from a tertiary university hospital. Method The study conducted a descriptive analysis of a case series using data from the Unit of Pathological Anatomy Laboratory and the Center for Infectious Diseases between 1999 and 2020. Results Four male patients, with a mean age of 32 years (ranging from 23 to 46), were evaluated. Key symptoms reported during medical consultations at the Urology outpatient service included scrotal nodules, increased testicular volume, and orchialgia, with an average symptom duration of six months. Physical examinations revealed a hard nodular mass in the epididymis or upper testicular pole, mostly on the right side (in half of the cases) or the left side (in the other half). Histological examination of biopsies indicated chronic granulomatous inflammation consistent with tuberculoid in all cases, with negative tumor markers. Notably, none of the patients reported contact with tuberculosis or leprosy patients, and only one patient had a positive PPD test (>24mm). Conclusion Genitourinary tuberculosis is a treatable and preventable disease. Understanding its clinical aspects, forms of presentation, dissemination, diagnosis, and treatment is crucial. Drug therapy, including rifampicin, isoniazid, pyrazinamide, and ethambutol, was effective in resolving symptoms in all patients. Surgical intervention in the form of orchidectomy was required for some cases, resulting in positive outcomes. Financing No conflict.

Abstract 291: Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types

Abstract Background: Stromal cells, including CAFs, are a heterogeneous group of cells in the tumor microenvironment. Many models describe their heterogeneity like the classification into 3 main subtypes in breast cancer: myCAF (myofibroblasts), iCAF (inflammatory CAF), and PVL (perivascular-like) cells. However, a pan-cancer model has not been established to exhaustively characterize stromal cell heterogeneity, undermining efforts in targeting CAFs with anti-tumor therapies. We investigated the heterogeneity of stromal cells in diverse diagnoses with single-cell RNA sequencing (scRNA-seq) data and bulk RNA-seq of sorted stromal cells to define consistent subtypes. Design: First, a meta-cohort of 11 scRNA-seq open-source datasets (>500,000 cells, >100 patients, 6 diagnoses) was analyzed. We also generated a scRNA-seq dataset of CD90+ cells derived from tumor tissue of an ovarian cancer patient. Additionally, bulk RNA-seq of 6 PVL, 2 iCAF, and 4 myCAF sorted cell samples across four solid cancers was performed. scRNA-seq data was analyzed with the SCANPY Python package. Results: Stromal cell clusters were defined as positive for PDGFRB, THY1 (CD90), and COL1A1 expression, and negative for the expression of known non-stromal and endothelial cell markers, by separately analyzing the open-source scRNA-seq datasets. Analysis of stromal cell heterogeneity revealed that the CAFs were consistently classified into 3 subtypes across all datasets: myCAF (high expression: FAP and ACTA2), PVL (high expression: MCAM (CD146) and ACTA2), and iCAF (high expression: APOD and cytokines IL6, CXCL12, CXCL2). Differential gene expression analysis of the scRNA-seq meta-cohort allowed us to develop functional gene expression signatures to define each subtype. All scRNA-sequenced CD90+ cells from ovarian cancer tissue were classified into the CAF subtypes. The myCAF and PVL signatures were specifically upregulated in the corresponding cell types (p<0.01, Mann-Whitney U test). While the iCAF signature was enriched in both iCAFs and myCAFs, myCAF and PVL signatures were downregulated in iCAFs. Finally, we developed a fluorescence-activated cell sorting (FACS) panel to separate CAF subtypes, using negative markers (CD45, CD31, EPCAM), a positive marker for stromal cells (CD90) and cell-type surface markers (CD146, FAP, CD91, CD44) for myCAF, iCAF and PVL cells. The high expression of CAF-specific genes in the sorted CAF populations denoted high sample purity. Importantly, the 3 CAF subpopulations separated by gating based on different levels of cell-type surface markers showed expression patterns congruent with the developed signatures across diagnoses. Summary: Altogether, the consistent classification of stromal cells with our cell-specific expression profiles demonstrated that the 3 CAF subtypes are conserved in many cancer types, offering groundwork for a pan-cancer model of stromal cell heterogeneity. Citation Format: Andrey Kravets, Boris Shpak, Anastasia Zotova, Maria Savchenko, Sofya Kust, Olga Golubeva, Mary Abdou, Fedor Grigoryev, Irina Bulusheva, Beniamin Sargsyan, Polina Pavlovich, Vlad Maximov, Vladimir Kushnarev, Michael Goldberg, Nikita Kotlov, Alexander Bagaev. Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 291.

Isolation, Biological Characterization, and Quality Inspection of Human Umbilical Cord-derived Mesenchymal Stem Cells for Clinical Research

Background: Human umbilical cord mesenchymal stem cells (hUCMSCs) represent a valuable and versatile cell type derived from the umbilical cord tissue of newborns. These cells exhibit diverse characteristics, including multipotent differentiation potential and low immunogenicity. hUCMSCs hold promise for clinical applications, yet concerns persist regarding their chromosomal stability. This study employs short tandem repeat (STR) analysis to assess genomic integrity. By focusing on chromosomal stability, the study aims to enhance confidence in the clinical viability of hUCMSCs, contributing to their safe and effective use in regenerative medicine. hUCMSCs were extracted from healthy newborn umbilical cords using tissue block adherence and enzyme digestion. The third generation of hUCMSCs undergoes biological characterization and quality checks, including examinations of cell morphology, viability, growth curves, surface markers, cell cycle, and multi-lineage differentiation potential. The result of the present study shows that hUCMSCs exhibit mesenchymal stem cell characteristics, with robust growth and high viability. Positive surface markers are expressed at rates exceeding 95%, while negative markers are ≤2%. Cell karyotyping analysis, STR spectrum identification, and chromosomal microarray analysis confirm genetic stability. Microbial testing shows the absence of contaminants, and immunological studies demonstrate the immunomodulatory capabilities of hUCMSCs. Specific residues, such as trypsin, were not detected. In conclusion, hUCMSCs produced under strict GLP and GMP conditions meet the quality guidelines for clinical application in various stem cell therapies. The findings support the clinical application of hUCMSCs in various medical contexts.

Transforming Growth Factor Beta 2 (TGFB2) mRNA Levels, in Conjunction with Interferon-Gamma Receptor Activation of Interferon Regulatory Factor 5 (IRF5) and Expression of CD276/B7-H3, Are Therapeutically Targetable Negative Prognostic Markers in Low-Grade Gliomas.

LGG tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. We conducted a study analyzing mRNA expression datasets from the UCSC Xena web platform. To screen for upregulated genes, we sought to compare normal brain tissue with LGG tumor samples. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Three tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (p < 0.0001), 8.9-fold (p < 0.0001), and 15.6-fold increase in mRNA expression levels, respectively, in LGG tumors. In addition, both TGFB1 (4.1-fold increase, p < 0.0001) and TGFB2 (2.2-fold increase, p < 0.0001) ligands were also upregulated in these tumors compared to normal brain tissue, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive, angiogenic, and pro-tumorigenic TME in gliomas mediated through TAMs. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and the downstream signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. Interestingly, the mRNA expression of a tumor-associated antigen, CD276/B7-H3, showed a significant (p < 0.0001) 4.03-fold increase in tumor tissue, giving further insights into the roles of macrophages and tumor cells in supporting the immunosuppressive TME. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25th percentile cut-off) of TGFB2 was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5, respectively) and age at diagnosis. Patients with high levels of TGFB2 and IFNGR2 were over-represented by LGG patients with isocitrate dehydrogenase wild-type (IDHwt) mutation status. The prognostic impact of high levels of TGFB2 and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival, suggesting that risk stratification of patients identifies LGG patients with IDH wild-type and high levels of TGFB2 in the design of clinical trials. Furthermore, we have additional IRF5 and CD276/B7-H3 as prognostic markers that can also be targeted for combination therapies with TGFB2 inhibitors. In support of these findings, we demonstrated that low levels of gene methylation in TGFB2, IFNGR2, IRF1, IRF5, STAT1, and CD276 were associated with significantly worse overall survival (OS) outcomes. This suggests that potential mechanisms to increase the expression of these prognostic markers occur via the action of demethylation enzymes.

Prognostic marker CD27 and its micro-environmental in multiple myeloma

BackgroundThe Cluster of Differentiation 27 (CD27) is aberrantly expressed in multiple myeloma (MM) -derived. This expression facilitates the interaction between tumor and immune cells within TME via the CD27-CD70 pathway, resulting in immune evasion and subsequent tumor progression. The objective of this study is to investigate the correlation between CD27 expression and the prognosis of MM, and to elucidate its potential relationship with the immune microenvironment.MethodsIn this research, CD27 expression in T cells within the 82 newly diagnosed MM microenvironment was assessed via flow cytometry. We then examined the association between CD27 expression levels and patient survival. Subsequent a series of bioinformatics and in vitro experiments were conducted to reveal the role of CD27 in MM.ResultsClinical evidence suggests that elevated CD27 expression in T cells within the bone marrow serves as a negative prognostic marker for MM survival. Data analysis from the GEO database has demonstrated a strong association between MM-derived CD27 and the immune response, as well as the hematopoietic system. Importantly, patients with elevated levels of CD27 expression were also found to have an increased presence of MDSCs and macrophages in the bone marrow microenvironment. Furthermore, the PERK-ATF4 signaling pathway has been implicated in mediating the effects of CD27 in MM.ConclusionsWe revealed that CD27 expression levels serve as an indicative marker for the prognosis of MM patients. The CD27- PERK-ATF4 is a promising target for the treatment of MM.

The ‘Still Not’ Present in Andi: identifying the grammaticalization source

Abstract Andi (East Caucasian) features a verb form that marks an event that is (still) not happening, contrary to the speaker’s expectation (i.e. ‘Still Not’ Present). This form is unusual for several reasons. First, forms of this kind are not typical for the language family. Second, while it conveys negative semantics, the form does not contain negation marking, even though segmentally it is rather heavy in comparison to other synthetic tenses. Third, to our knowledge grammatical ‘still not’ expressions are typologically uncommon. The paper describes the semantics and morphosyntactic properties of the ‘Still Not’ Present and suggests a plausible grammaticalization source by comparing dialects and closely related languages. We also try to match the data to typological categories like phasal polarity.

Clinical characteristics and predictive factors of delayed diagnosis in patients with sellar germ cell tumors.

To investigate the clinical characteristics and predictive factors associated with delayed diagnosis in patients with sellar germ cell tumors (GCTs), aiming for early diagnosis. A total of 345 patients with sellar GCTs were retrospectively collected. Patients were classified into a delayed diagnosis group (> 6 months from onset to diagnosis) and a non-delayed diagnosis group (≤ 6 months). We compared general characteristics, clinical symptoms, diagnostic methods, treatment strategies, tumor prognosis, and pituitary function between the two groups. Predictive factors for delayed diagnosis were explored using multivariate logistic regression analysis. 225 patients (65.2%) experienced delayed diagnosis. Although there was no association between delayed diagnosis and survival rates or tumor recurrence rates, the delayed diagnosis group had a higher incidence of central diabetes insipidus, central adrenal insufficiency, central hypothyroidism, central hypogonadism, and growth hormone deficiency. Moreover, polyuria/polydipsia (OR 5.46; 95% CI 2.33-12.81), slow growth (OR 5.86; 95% CI 2.61-13.14), amenorrhea (OR 6.82; 95% CI 2.68-17.37), and germinoma (OR 4.99; 95% CI 1.08-3.61) were associated with a higher risk of delayed diagnosis, while older age of onset (OR 0.88; 95% CI 0.84-0.94) and nausea/vomiting (OR 0.31; 95% CI 0.15-0.63) contributed to earlier diagnosis. In patients with sellar GCTs, delayed diagnosis is common and linked to increased pituitary dysfunction. The initial symptoms of slow growth, polyuria/polydipsia, and amenorrhea, as well as germinoma with negative tumor markers, predict the possibility of a delayed diagnosis. Early diagnosis is crucial to minimize the impact of sellar GCTs on pituitary function.

Changing Etiological Spectrum of Hepatocellular Carcinoma in India—A Systematic Review and Meta-analysis

BackgroundRecent studies from both India and outside India have shown a change in the etiological profile of hepatocellular carcinoma (HCC). We aimed to analyze the etiological spectrum and changing trends of HCC etiology in India using a systematic review of current literature and meta-analysis. MethodsElectronic databases of Pubmed/Medline, Scopus, and Embase were searched from inception to July 2023 for studies reporting the data on the etiology of HCC from India. The pooled proportions with 95% confidence interval were calculated using summative statistics. ResultsA total of 60 studies (n = 12327) were included in the final analysis. The pooled proportions of HCC cases with at least one positive and negative viral marker were 56.0 (49.5 – 62.6) and 43.1% (36.5 – 49.8), respectively. The pooled proportion of HCC cases with positive hepatitis B virus (HBV) markers was 41.0 (35.8 – 46.1), while those with positive markers for hepatitis C virus (HCV) were 20.3 (17.0 – 23.6). The pooled proportion of cases with HCC with significant alcohol intake was 19.0% (15.6 – 22.4), and those related to non-alcoholic fatty liver disease (NAFLD) were 16.9% (12.1 – 21.7). Around 7.9% (5.8 – 10.0) of the cases had HCC with multiple etiologies. Subgroup analysis showed a significant variation with the location of the study based on zone. Meta-regression analysis based on publication year (1990-2023) showed a significant reduction in the proportion of cases with HBV and an increase in cases with NAFLD. In contrast, the proportion of cases with HCV and alcohol did not change significantly. ConclusionViral hepatitis is the most common etiology of HCC in India, predominantly HBV. The proportions of cases with HCC related to NAFLD are increasing, and those related to HBV are declining.

Disentangling inhibition and prediction in negation processing

Negation as a universal feature of human language is used effortlessly in everyday communication. However, experimental research has shown that the comprehension of negated sentences seems to require additional cognitive resources compared to affirmative sentences. Many studies investigating the processing of negation report longer reading and reaction times for negative compared to affirmative sentences and many studies report a Polarity by Truth interaction: false affirmative sentences lead to longer response times and larger N400 event-related potentials (ERPs) than true affirmative sentences, whereas the pattern is reversed for negative sentences where it is the true sentence that elicits longer reaction times and higher N400 ERPs compared to false negative sentences. These interactions have been discussed in the light of lexical associations, predictability, and the need to construct two subsequent mental representations. Furthermore, recent studies have shown that the comprehension of negated sentences seems to make use of neural resources that are typically involved in cognitive control and inhibitory mechanisms. As both processes have been associated with two different and temporally overlapping ERP components (the N400 and the P300), we focus on studies with high temporal resolution. We discuss linguistic aspects of negation, such as semantic similarity and contextual invariance of negation. We furthermore discuss the role of the verb as well as the position of the negative marker with respect to the verb, and their respective relevance for predictive and inhibitory mechanisms in negated sentences.

Identification and prognostic analysis of ferroptosis‑related gene HSPA5 to predict the progression of lung squamous cell carcinoma.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, is implicated in the development and therapeutic responses of cancer. However, the role of ferroptosis-related gene profiles in lung squamous cell carcinoma (LSCC) remains largely unknown. The present study aimed to identify the prognostic roles of ferroptosis-related genes in LSCC. Sequencing data from the Cancer Genome Atlas were analyzed and ferroptosis-related gene expression between tumor and para-tumor tissue was identified. The prognostic role of these genes was also assessed using Kaplan-Meier analyses and univariate and multivariate Cox proportional hazards regression model analyses. Immunological correlation, tumor stemness, drug sensitivity and the transcriptional differences of heat shock protein (HSP)A5 in LSCC were also analyzed. Thereafter, the expression of HSPA5 in 100 patients with metastatic LSCC was evaluated using immunohistochemistry (IHC) and the clinical significance of these markers with different risk factors was assessed. Of the 22 ferroptosis-related genes, the expression of HSPA5, HSPB1, glutathione peroxidase 4, Fanconi anemia complementation group D2, CDGSH iron sulfur domain 1, farnesyl-diphosphate farnesyltransferase 1, nuclear factor erythroid 2 like 2, solute carrier (SLC)1A5, ribosomal protein L8, nuclear receptor coactivator 4, transferrin receptor and SLC7A11 was significantly increased in LSCC compared with adjacent tissues. However, only high expression of HSPA5 was able to predict progression-free survival (PFS) and disease-free survival in LSCC. Although HSPA5 was also significantly elevated in patients with lung adenocarcinoma, HSPA5 expression did not predict the prognosis of patients with lung adenocarcinoma. Of note, a higher expression of HSPA5 was related to higher responses to chemotherapy but not to immunotherapy. In addition, HSPA5 expression was positively correlated with 'ferroptosis', 'cellular responses to hypoxia', 'tumor proliferation signature', 'G2M checkpoint', 'MYC targets' and 'TGFB'. IHC analysis also demonstrated that a high expression of HSPA5 in patients with metastatic LSCC in the study cohort was associated with shorter PFS and overall survival. In conclusion, the present study demonstrated that the expression of the ferroptosis-related gene HSPA5 may be a negative prognostic marker for LSCC.

Experience of humanistic nursing in hemodialysis nursing for patients with diabetic kidney disease.

Diabetic kidney disease (DKD) is a prevalent complication of diabetes that often requires hemodialysis for treatment. In the field of nursing, there is a growing recognition of the importance of humanistic care, which focuses on the holistic needs of patients, including their emotional, psychological, and social well-being. However, the application of humanistic nursing in the context of hemodialysis for DKD patients remains relatively unexplored. To explore the experience of humanistic nursing in hemodialysis nursing for DKD patients. Ninety-six DKD patients treated with hemodialysis from March 2020 to June 2022 were included in the study and divided into the control cluster (48 cases) and the study cluster (48 cases) according to different nursing methods; the control cluster was given routine nursing and the study cluster was given humanized nursing. The variances of negative emotion mark, blood glucose, renal function, the incidence of complications, life mark and nursing satisfaction before and after nur-sing were contrasted between the two clusters. No significant difference in negative emotion markers between the two clusters were observed before nursing (P > 0.05), and the negative emotion markers of the two clusters decreased after nursing. The Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale markers were lower in the study cluster than the control cluster. The healing rate of patients in the study cluster was significantly higher than the control cluster (97.92% vs 85.42%, P < 0.05). Blood glucose parameters were not significantly different between the groups prior to nursing (P > 0.05). However, after nursing, blood urea nitrogen and serum creatinine (SCr) levels in the study cluster were lower than those in the control cluster (P < 0.05). The incidence rate of complications was significantly lower in the study group compared to the control cluster (6.25% vs 20.83%, P < 0.05). There was no significant difference in the life markers between the two clusters before nursing. While the life markers increased after nursing for both groups, the 36-item health scale markers in the study cluster were higher than those within the control cluster (P < 0.05). Finally, the nursing satisfaction rate was 93.75% in the study cluster, compared to 75% in the control cluster (P < 0.05). In hemodialysis for DKD patients, the implementation of humanistic nursing achieved ideal results, effectively reducing patients' psychological negative emotion markers so that they can actively cooperate with the diagnosis and nursing, facilitate the control of blood glucose and the maintenance of residual renal function, reduce the occurrence of complications, and finally enhance the life quality and nursing satisfaction of patients. It is worthy of being widely popularized and applied.

Evaluation of stability and safety of equine mesenchymal stem cells derived from amniotic fluid for clinical application.

Amniotic fluid mesenchymal stem cells (AF-MSCs), which can be obtained from fetal tissue, reportedly have self-renewal capacity and multi-lineage differentiation potential. The aim of this study was to identify the biological characteristics of AF-MSCs and evaluate their stability and safety in long-term culture. To confirm the biological characteristics of AF-MSCs, morphology, proliferation capacity, karyotype, differentiation capacity, gene expression level, and immunophenotype were analyzed after isolating AF-MSCs from equine amniotic fluid. AF-MSCs were differentiated into adipocytes, chondrocytes, and osteocytes. Immunophenotype analyses revealed expression levels of ≥95% and ≤ 2% of cells for a positive and negative marker, respectively. Analysis of the MSCs relative gene expression levels of AF-MSCs was approximately at least twice that of the control. The endotoxin level was measured to verify the safety of AF-MSCs and was found to be less than the standard value of 0.5 EU/ml. AF-MSCs were cultured for a long time without any evidence of abnormalities in morphology, proliferation ability, and karyotype. These results suggest that amniotic fluid is a competent source for acquiring equine MSCs and that it is valuable as a cell therapy due to its high stability.

Initial surveillance in men with marker negative clinical stage IIA non-seminomatous germ cell tumours.

To assess whether extended surveillance with repeated computed tomography (CT) scans for patients with clinical stage IIA (CS IIA; <2 cm abdominal node involvement) and negative markers (Mk-) non-seminomatous germ cell tumours (NSGCTs) can identify those with true CS I. To assess the rate of benign lymph nodes, teratoma, and viable cancer in retroperitoneal lymph node dissection (RPLND) histopathology for patients with CS IIA Mk- NSGCT. Observational prospective population-based study of patients diagnosed 2008-2019 with CS IIA Mk- NSGCT in the Swedish and Norwegian Testicular Cancer Group (SWENOTECA) registry. Patients were managed with surveillance, with CT scans, and tumour markers every sixth week for a maximum of 18 weeks. Patients with radiological regression were treated as CS I, if progression with chemotherapy, and remaining CS IIA Mk- disease with RPLND. The end-point was the number and percentage of patients down-staged to CS I on surveillance and rate of RPLND histopathology presented as benign, teratoma, or viable cancer. Overall, 126 patients with CS IIA Mk- NSGCT were included but 41 received therapy upfront. After surveillance for a median (range) of 6 (6-18) weeks, 23/85 (27%) patients were in true CS I and four (5%) progressed. Of the remaining 58 patients with lasting CS IIA Mk- NSGCT, 16 received chemotherapy and 42 underwent RPLND. The RPLND histopathology revealed benign lymph nodes in 11 (26%), teratoma in two (6%), and viable cancer in 29 (70%) patients. Surveillance with repeated CT scans can identify patients in true CS I, thus avoiding overtreatment. The RPLND histopathology in patients with CS IIA Mk- NSGCT had a high rate of cancer and a low rate of teratoma.

Efficient induction of pluripotent stem cells differentiated into mesenchymal stem cell lineages.

Mesenchymal stem cells (MSCs) have garnered significant attention in the field of cell-based therapy owing to their remarkable capabilities for differentiation and self-renewal. However, primary tissue-derived MSCs are plagued by various limitations, including constrained tissue sources, arduous and invasive retrieval procedures, heterogeneous cell populations, diminished purity, cellular senescence, and a decline in self-renewal and proliferative capacities after extended expansion. Addressing these challenges, our study focuses on establishing a robust differentiation platform to generate mesenchymal stem cells derived from induced pluripotent stem cells (iMSCs). To achieve this, we used a comprehensive methodology involving the differentiation of induced pluripotent stem cells into MSCss. The process was meticulously designed to ensure the expression of key MSC positive markers (CD73, CD90, and CD105) at elevated levels, coupled with the minimal expression of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Moreover, the stability of these characteristics was evaluated across 10th generations. Our findings attest to the success of this endeavor. iMSCs exhibited robust expression of positive markers and limited expression of negative markers, confirming their MSC identity. Importantly, these characteristics remained stable even up to the 10th generation, signifying the potential for sustained use in therapeutic applications. Furthermore, our study demonstrated the successful differentiation of iMSCs into osteocytes, chondrocytes, and adipocytes, showcasing their multilineage potential. In conclusion, the establishment of induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) presents a significant advancement in overcoming the limitations associated with primary tissue-derived MSCs. The remarkable stability and multilineage differentiation potential exhibited by iMSCs offer a strong foundation for their application in regenerative medicine and tissue engineering. This breakthrough paves the way for further research and development in harnessing the full therapeutic potential of iMSCs.

Establishing the role of BRCA1 in the diagnosis, prognosis and immune infiltrates of breast invasive cancer by bioinformatics analysis and experimental validation.

Breast cancer susceptibility gene 1 (BRCA1) is a well-known gene that acts a vital role in suppressing the growth of tumors. Previous studies have primarily focused on the genetic mutations of BRCA1 and its association with hereditary breast invasive carcinoma (BRCA). However, little research has been done to investigate the relationship between BRCA1 and immune infiltrates and prognosis in BRCA. We obtained the expression profiles and clinical information of patients with BRCA from the Cancer Genome Atlas (TCGA) database. The levels of the BRCA1 gene between BRCA tissues and normal breast tissues were compared through the Wilcoxon rank-sum test. Additionally, we performed WB and RT-qPCR techniques to detect the expression of BRCA1. We conducted functional enrichment analyses. Furthermore, we assessed immune cell infiltration using a single-sample gene set enrichment analysis. The methylation status of the BRCA1 gene was analyzed using the UALCAN and MethSurv databases. The Cox regression analysis and (KM) Kaplan-Meier method were employed to determine the prognostic value of BRCA1. In order to provide a practical tool for predicting the overall survival rates at different time points, we also constructed a nomogram. Our analysis revealed that the expression of BRCA1 was significantly higher in BRCA tissues compared to normal tissues. Furthermore, this increased level of BRCA1 was found to be associated with specific BRCA subtypes, including T2, stage II, ER positive, ect. Importantly, the overexpression of BRCA1 was shown to be a negative prognostic marker for the overall survival rates of BRCA patients. Moreover, low methylation status of the BRCA1 gene was related to a poorer prognosis. Furthermore, our results indicated that high levels of BRCA1 are related to a decrease in level of killer immune cells, such as natural killer (NK) cells, macrophages, CD8+ T cells, and plasma-like dendritic cells (pDCs) within the tumor microenvironment. Our study is the first to provide evidence indicating that the presence of BRCA1 can serve as a reliable marker for both diagnosing and determining the prognosis of BRCA. Moreover, BRCA1 acts as a crucial indicator of the cancer's potential to infiltrate and invade the immune system, which has important implications for developing targeted therapies in BRCA.

P-05 Malignant pheochromocytoma with a negative biomarker hormonal assay: A case report

Abstract Description Malignant pheochromocytoma is a rare, predominantly sporadic, but also familial disease, with a malignancy frequency of about 10%. Clinical Case A 25-year-old male was referred to us by the general surgeon after he had a hypertensive crisis during the appendectomy surgery and then accidentally discovered a left adrenal gland mass by CT scan. Assessment of the patient done by physical examination, the height 168cm, weight 53 kg and the BMI 19 kg/m2 and the vital sign was Bp 200/110, and PR 87 b/min. Investigations: normal renal function test and negative metanephrines, normetanephrine and aldosterone, renin and aldosterone renin ratio also normal serum electrolytes. Imaging abdomen CT shows a mass measuring 4.8 X3.7X6.2 cm in its (AP, TS&amp;CC dimension) in the left adrenal gland (encircle), density 40-50 HU. with central hypo density represented cystic necrosis. Right adrenal is normal limbs following intravenous contrast, the left adrenal mass enhances brightly (130-140HU) with central cystic necrosis … consisting with typical features of pheochromocytoma. The patient referred to expert surgeon and there was some preoperative preparation inform of the patient received phenoxybenzamine (alpha blocker) the receive propranolol to control reflux tachycardia and normalized the blood pressure, resection of left adrenal mass was done and the gross tissue biopsy was sent to histopathologist, and the result revealed a diagnosis of malignant pheochromocytoma, after two weeks from operation CT images post operatively after total resection of left adrenal mass revealed clear bed no residual SOL with surgical clips seen. Conclusion Marker-negative Pheochromocytomas are an uncommon form of the illness. Even more dangerous forms, malignant pheochromocytomas typically release metabolites. However, a doctor must be alert to patients with negative markers who exhibit typical symptoms and must make a diagnosis using a combination of clinical, radiologic, and biochemical testing. When choosing patients for presurgical alpha blockade, this is extremely important. Table 1

Exploring chronic and transient tumor hypoxia for predicting the efficacy of hypoxia-activated pro-drugs

Hypoxia, a low level of oxygen in the tissue, arises due to an imbalance between the vascular oxygen supply and oxygen demand by the surrounding cells. Typically, hypoxia is viewed as a negative marker of patients’ survival, because of its implication in the development of aggressive tumors and tumor resistance. Several drugs that specifically target the hypoxic cells have been developed, providing an opportunity for exploiting hypoxia to improve cancer treatment. Here, we consider combinations of hypoxia-activated pro-drugs (HAPs) and two compounds that transiently increase intratumoral hypoxia: a vasodilator and a metabolic sensitizer. To effectively design treatment protocols with multiple compounds we used mathematical micro-pharmacology modeling and determined treatment schedules that take advantage of heterogeneous and dynamically changing oxygenation in tumor tissue. Our model was based on data from murine pancreatic cancers treated with evofosfamide (as a HAP) and either hydralazine (as a vasodilator), or pyruvate (as a metabolic sensitizer). Subsequently, this model was used to identify optimal schedules for different treatment combinations. Our simulations showed that schedules of HAPs with the vasodilator had a bimodal distribution, while HAPs with the sensitizer showed an elongated plateau. All schedules were more successful than HAP monotherapy. The three-compound combination had three local optima, depending on the HAPs clearance from the tissue interstitium, each two-fold more effective than baseline HAP treatment. Our study indicates that the three-compound therapy administered in the defined order will improve cancer response and that designing complex schedules could benefit from the use of mathematical modeling.

‘Until’ constructions and expletive negation in Huasteca Nahuatl

Abstract A number of works have explored expletive negation in clause-linkage constructions. Most of them have shown that this type of negative marker can be omitted from the adverbial clause without affecting the interpretation holding between clauses. The study shows, based on the analysis of natural discourse data, that expletive negation has developed an intriguing discourse function in three types of ‘until’ constructions in Huasteca Nahuatl: ‘not…until’ constructions, scalar additive ‘until’ constructions, and beginning-to-end constructions. When the negative marker amo appears in the ‘until’ clause, the proposition should be characterized as expressing surprise. When it is absent from the ‘until’ clause, the proposition does not express surprise. This function of the expletive negative marker amo does not appear in elicited sentences, but only in spontaneous speech. It is proposed that Huasteca Nahuatl developed expletive negation in ‘not…until’ constructions due to contact with Spanish. However, the development of expletive negation in scalar additive ‘until’ constructions and beginning-to-end constructions is an internally motivated development in Huasteca Nahuatl that cannot be attributed to Spanish.

Expression and Clinical Significance of CD30 and CD56 in Lymphoblastic Lymphoma: A Retrospective Analysis on Paraffin-Embedded Tissues by Immunohistochemistry

Background: We evaluated CD30 and CD56 expression in lymphoblastic lymphoma (LBL) and correlated the results with clinicopathological features and prognosis. Methods: Immunohistochemical (IHC) staining was performed on 85 formalin-fixed paraffin-embedded LBL specimens using two CD30 clones and one CD56 antibody clone. Results: Weak and diffuse expression of CD30 was expressed in 4.7% (clone Ber-H2) or 14.1% (clone EPR4102) in LBL, while CD56 was expressed in 24.7%. CD30 and CD56 expression correlated with lactate dehydrogenase levels. CD56-positive expression was closely associated with an unfavorable prognosis. Although CD30 expression exhibited a trend toward poorer overall survival, it did not reach statistical significance. Conclusion: CD56 is a potential negative prognostic marker. These findings suggest that CD30 and CD56 targeted therapies could be potential therapeutic targets for LBL patients.

Celiac disease diagnosis: transglutaminase, duodenal biopsy and genetic tests correlations.

Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion in genetically susceptible individuals. The haplotypes HLA-DQ2 and DQ8, transglutaminase (TGA) antibodies, and biopsy findings are the main tests performed in the evaluation and CD diagnosis. The objective was to establish possible correlations between transglutaminase levels, genetic markers tests, and qualitative intestinal biopsy findings (modified Marsh classification) at the diagnosis. A retrospective cohort study. The selection criteria were confirmed CD cases with genetic tests performed. Statistical analysis was done mainly through One-way ANOVA, Kendall's correlation coefficient (T), and linear regression. The study included 112 patients, with a mean age of 6 ± 4 years. All cases were tested to HLA-DQ2, and it was positive in 93%. HLA-DQ8 was tested in 73% of cases and it was positive in 61%. The percentage of negative genetic markers (DQ2/DQ8) was 4.5% for patients tested to both haplotypes. A comparison of DQ2/DQ8 (positive and negative) with clinical findings and tests performed did not identify any differences for most of the parameters analyzed. Cases of type I diabetes presented significant negative expression for DQ2(-); p = 0.05 and positive expression for DQ8(+); p = 0.023. The TGA antibody levels ranged from 18 to 36,745 U/ml. An inverse correlation was found between age and TGA-L level (p = 0.043). In 23% of the cases, the TGA levels were greater than 1,000 U/ml and presented a moderate positive correlation with the atrophy biopsy profile (T = 0.245). Patients with an atrophic biopsy profile (Marsh III) had a moderate positive correlation with growth failure (T = 0.218) but a negative correlation with constipation (T = -0.277). In terms of diagnosis tests for CD, transglutaminase levels and age presented an inverse correlation, with the level decreasing as age increased. A moderately positive correlation was found between mean transglutaminase with intestinal atrophy and growth retardation. The genetic test DQ2 was positive for 93% and negative genetic markers (DQ2/DQ8) represented 4.5% of cases studied.