Search Results (153)

Background/Objectives: Neutrophils have recently gained significant attention due to their heterogeneity in tumor settings. Recent data showed neutrophil pro- and anti-tumor profiles during tumor progression. However, the concessive causes of neutrophil skewing toward one or another profile are not fully understood. Methods: In this study, using RT-qPCR, flowcytometry, and confocal microscopy, we investigated the phenotype of splenic neutrophils of mice bearing Lewis lung carcinoma LLC, RLS₄₀ lymphosarcoma, and B16 melanoma. Results: Our data showed an immunosuppressive phenotype in the case of the LLC model with PD-L1 and IL10 expression. In the B16 model, minimal changes in the neutrophil phenotype were observed, regardless of tumor size. In the RLS₄₀ model, the neutrophil phenotype was associated with the tumor growth rate, where, in aggressively progressed tumors (RLS₄₀^High), CCL17 was expressed, while, in mice with controlled tumor growth (RLS₄₀^Low), anti-tumor markers were expressed (FAS, ICAM-1, PD-L1). DNase I treatment significantly reduced tumor growth and metastasis in the RLS₄₀ model but not in B16, enhanced the anti-tumor profile in RLS₄₀ neutrophils, and tended to reduce NET formation induced by A23187. Conclusions: The phenotype of neutrophils from tumor-bearing mice is influenced by the tumor type and progression stage. DNase I had anti-tumor, antimetastatic, and immunostimulatory effects and significantly modified the neutrophil profile in the immunogenic model RLS₄₀. Full article

Measles, hepatitis C, and COVID-19 are significant human diseases caused by RNA viruses. While vaccines exist to prevent infections, there are a small number of currently available therapeutic agents that can effectively treat these diseases after infection occurs. This study explores a new therapeutic strategy using a small molecule designated polyprenyl immunostimulant (PI) to increase innate immune responses and combat viral infections. Using a multi-disciplinary approach, this study quantifies the effects of PI in mice and THP-1 cells using flow cytometry to identify immune phenotypic markers and mass spectroscopy to monitor the metabolomic profiles of immune cells perturbed by PI treatment. The metabolomic studies identified that sphinganine and ceramide, which are precursors of sphingosine-1-phosphate (S1P), were the common metabolites upregulated in THP-1 and mice blood. Sphingosine-1-phosphate can mediate the trafficking of T cells, whereas ceramide can signal the activation and proliferation of T cells, thereby modulating the mammalian host’s immunity. To demonstrate proof-of-principle, a case study was conducted to examine the benefit of administering PI to improve the outcomes of a feline co-infected with two distinct RNA viruses—feline leukemia virus and feline infectious peritonitis virus. Both viruses produce deadly symptoms that closely resemble RNA viruses that infect humans. The results identify quantifiable cellular and metabolic markers arising from PI treatment that can be used to establish a platform measuring the efficacy of PI in modulating the innate immune system. Full article

Cell-mediated immunity is an important component of the immune response for intracellular pathogens. Live vaccines containing different pathogens are used concurrently in the field but are generally licensed individually. Concurrent administration of these vaccines has led to concerns about immune interference. The goal of this study was to characterize BVDV-specific responses to vaccination and determine the effect of concurrent Brucella abortus strain RB51 (RB51) vaccine administration. Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with a modified-live viral (MLV) vaccine containing BVDV, both RB51 and an MLV, or unvaccinated controls were utilized to evaluate antibody titers and the frequency of interferon gamma (IFN-γ) production within CD4⁺, CD8⁺ T cells, and NK cell populations via flow-cytometry. Our data demonstrated the lack of vaccine interference following concurrent administration of two common bovine MLVs and may even suggest some level of enhanced IFN-γ production with concurrent administration. Full article

The etiology of acute myeloid leukemia (AML) is complex, including genetic and environmental abnormalities. The immune system anomalies play an essential role in the process of leukemogenesis. However, the immunopathological factors, including abnormal T helper (Th) subsets, contributing to the initiation and progression of this neoplasm, require further investigation. Considering the previously mentioned data, we decided to study the expression pattern of transcription factors T-bet, Foxp3, and RORγt that regulate Th1, Treg, and Th17, respectively, in acute myeloid leukemia with correlation to clinical and other investigation data and treatment outcomes. This study was conducted on 80 newly diagnosed patients with AML recruited from the National Cancer Institute, Cairo University, and 25 healthy control subjects. The AML patient cohort consisted of 30 females (37.5%) and 50 males (62.5%), ranging from 18 to 74 years old. The control group was 8 females (32%) and 17 males (68%), with ages ranging from 23 to 40 years old. Samples were provided from the bone marrow of donor cases for allogeneic bone marrow transplantation. The diagnosis of acute myeloid leukemia was based on morphologic and cytochemical evaluation, immunophenotyping, and complementary cytogenetics according to WHO criteria. Upshift from the normal T-bet intensity of power (MFI), RORγt⁺ CD4⁺ T lymphocyte frequency (%) with downshift from the normal FOXP3 intensity of power (MFI), may suggest a state of inflammation. In contrast, an upshift from the normal FOXP3⁺ CD4⁺ T lymphocyte frequency (%) may reflect a state of immunosuppression in the bone marrow microenvironment of AML. Combined, they constitute a sophisticated scenario of immunological disorder in AML. Co-expression of T-bet and RORγt transcription factors in CD4⁺ T lymphocytes in both normal and AML groups may suggest CD4⁺ T lymphocyte plasticity. Full article

Background. Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia defined by the presence of a genetic abnormality, namely the PML::RARA gene fusion, as the result of a reciprocal balanced translocation between chromosome 17 and chromosome 15. APL is a veritable emergency in hematology due to the risk of early death and coagulopathy if left untreated; thus, a rapid diagnosis is needed in this hematological malignancy. Needless to say, cytogenetic and molecular biology techniques, i.e., fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR), are essential in the diagnosis and management of patients diagnosed with APL. In recent years, the use of artificial intelligence (AI) and its brances, machine learning (ML), and deep learning (DL) in the field of medicine, including hematology, has brought to light new avenues for research in the fields of blood cancers. However, to our knowledge, there is no comprehensive evaluation of the potential applications of AI, ML, and DL in APL. Thus, the aim of the current publication was to evaluate the prospective uses of these novel technologies in APL. Methods. We conducted a comprehensive literature search in PubMed/MEDLINE, SCOPUS, and Web of Science and identified 20 manuscripts eligible for the qualitative analysis. Results. The included publications highlight the potential applications of ML, DL, and other AI branches in the diagnosis, evaluation, and management of APL. The examined AI models were based on the use of routine biological parameters, cytomorphology, flow-cytometry and/or OMICS, and demonstrated excellent performance metrics: sensitivity, specificity, accuracy, AUROC, and others. Conclusions. AI can emerge as a relevant tool in the evaluation of APL cases and potentially contribute to more rapid screening and identification of this hematological emergency. Full article

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug’s commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug’s clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene’s functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay’s adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay’s specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug’s mechanism of action (MoA) in the same assay. Full article

Prostate cancer (PCa) is the tumour pathology with the second highest incidence among men worldwide. PCa is strongly influenced by obesity (OB), which increases its aggressiveness. Hence, some metabolic drugs like metformin have emerged as potential anti-tumour agents against several endocrine-related cancers. Likewise, a high adherence to the Mediterranean diet has been associated with lower rates of OB and a reduction in PCa aggressiveness since this diet contains phenolic bioactive compounds such as hydroxytyrosol (HT) that is mainly present in extra virgin olive oil. Thus, we decided to analyse the therapeutic potential of the combination of HT + metformin in different PCa cell models. Specifically, combinations of different doses of HT and metformin were evaluated by analysing the proliferation rate of LNCaP, 22Rv1, DU-145, and PC−3 cells using the SynergicFinder method. The results revealed a synergistic effect of HT + metformin in significantly reducing proliferation, especially in LNCaP cells. This anti-tumour effect of HT + metformin was also confirmed in migration and tumoursphere formation assays in LNCaP. The effects on the cell cycle and apoptosis were also assessed by flow-cytometry, and a cycle arrest in the G1 phase and an increase in late apoptosis were observed with the combination of HT + metformin. The phosphorylation levels of critical components of different oncogenic pathways were measured which revealed that the combination of HT + metformin significantly reduced the activity of multiple components of the MAPK, AKT, and TGF-β pathways. Overall, the combination of HT + metformin might represent a new therapeutic avenue for the management of PCa patients, an observation that certainly warrants further investigation through a well-designed clinical trial. Full article

Hydrocarbon fuel biofouling and biocorrosion require expensive cleanup of aviation infrastructures unless appropriate sustainment measures are applied. The identification of novel biological control agents offers promising alternatives to the current chemical biocides used in fuel sustainment. In this study, 496 microbial fuel isolates from our in-house repository were screened to identify new endogenously produced antimicrobial compounds. Using agar plug screening, liquid culture growth testing, and Jet A fuel culture assays, the two fuel-isolate strains Pseudomonas protegens #133, and Bacillus subtilis #232 demonstrated promising biocontrol activity against bacteria, yeast, and filamentous fungi. Liquid chromatography-quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS/MS) of #232 culture filtrate identified several common lipopeptide antimicrobials including gageostatin C, gageopeptin B, and miscellaneous macrolactins. In contrast, LC-QTOF-MS/MS identified the siderophore pyochelin as one of the predominant compounds in #133 culture filtrate with previously demonstrated antimicrobial effect. Jet fuel microbial consortium culture testing of #133 culture filtrate including flow-cytometry live/dead cell mechanism determination demonstrated antimicrobial action against Gram-positive bacteria. The study concludes that antimicrobial compounds secreted by #133 have bactericidal effects against Gordonia sp. and cause cell death through bacterial lysis and membrane damage with potential applications in the biocidal treatment of hydrocarbon-based aviation fuels. Full article

Background: Alpha radionuclide therapy has emerged as a promising novel strategy for cancer treatment; however, the therapeutic potential of ²²⁵Ac-labeled peptides in pancreatic cancer remains uninvestigated. Methods: In the cytotoxicity study, tumor cells were incubated with ²²⁵Ac-DOTA-RGD₂. DNA damage responses (γH2AX and 53BP1) were detected using flowcytometry or immunohistochemistry analysis. Biodistribution and therapeutic studies were carried out in BxPC-3-bearing mice. Results: ²²⁵Ac-DOTA-RGD₂ demonstrated potent cytotoxicity against cells expressing α_vβ₃ or α_vβ₆ integrins and induced G2/M arrest and γH2AX expression as a marker of double-stranded DNA breaks. ²²⁵Ac-DOTA-RGD₂ (20, 40, 65, or 90 kBq) showed favorable pharmacokinetics and remarkable tumor growth inhibition without severe side effects in the BxPC-3 mouse model. In vitro studies revealed that 5 and 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ swiftly induced G2/M arrest and elevated γH2AX expression. Furthermore, to clarify the mechanism of successful tumor growth inhibition for a long duration in vivo, we investigated whether short-term high radiation exposure enhances radiation sensitivity. Initially, a 4 h induction treatment with 5 and 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ enhanced both cytotoxicity and γH2AX expression with 0.5 kBq/mL of ²²⁵Ac-DOTA-RGD₂ compared to a treatment with only 0.5 kBq/mL of ²²⁵Ac-DOTA-RGD₂. Meanwhile, the γH2AX expression induced by 5 or 10 kBq/mL of ²²⁵Ac-DOTA-RGD₂ alone decreased over time. Conclusions: These findings highlight the potential of using ²²⁵Ac-DOTA-RGD₂ in the treatment of intractable pancreatic cancers, as its ability to induce G2/M cell cycle arrest enhances radiosensitization, resulting in notable growth inhibition. Full article

Our study highlights the apoptosis, cell cycle, DNA ploidy, and autophagy molecular mechanisms network to identify prostate pathogenesis and its prognostic role. Caspase 3/7 expressions, cell cycle, adhesion glycoproteins, autophagy, nuclear shrinkage, and oxidative stress by flow-cytometry analysis are used to study the BPH microenvironment’s heterogeneity. A high late apoptosis expression by caspases 3/7 activity represents an unfavorable prognostic biomarker, a dependent predictor factor for cell adhesion, growth inhibition by arrest in the G2/M phase, and oxidative stress processes network. The heterogeneous aggressive phenotype prostate adenoma primary cell cultures present a high S-phase category (>12%), with an increased risk of death or recurrence due to aneuploid status presence, representing an unfavorable prognostic biomarker, a dependent predictor factor for caspase 3/7 activity (late apoptosis and necrosis), and cell growth inhibition (G2/M arrest)-linked mechanisms. Increased integrin levels in heterogenous BPH cultures suggest epithelial–mesenchymal transition (EMT) that maintains an aggressive phenotype by escaping cell apoptosis, leading to the cell proliferation necessary in prostate cancer (PCa) development. As predictor biomarkers, the biological mechanisms network involved in apoptosis, the cell cycle, and autophagy help to establish patient prognostic survival or target cancer therapy development. Full article

Malignant mesothelioma is a rare tumor associated with asbestos exposure. Mesothelioma carcinogenesis is related to enhanced reactive oxygen species (ROS) production and iron overload. Despite the recent advances in biomedical sciences, to date the only available treatments include surgery in a small fraction of patients and platinum-based chemotherapy in combination with pemetrexed. In this view, the purpose of this study was to evaluate the therapeutic potential of the newly synthetized platinum prodrug Pt(IV)Ac-POA compared to cisplatin (CDDP) on human biphasic mesothelioma cell line MSTO-211H using different complementary techniques, such as flow-cytometry, transmission electron microscopy (TEM), and immunocytochemistry. Healthy mesothelial cell lines Met-5A were also employed to assess the cytotoxicity of the above-mentioned compounds. Our in vitro results showed that Pt(IV)Ac-POA significantly interfere with iron metabolisms and more importantly is able to trigger cell death, through different pathways, including ferroptosis, necroptosis, and apoptosis, in neoplastic cells. On the other hand, CDDP triggers mainly apoptotic and necrotic cell death. In conclusion, Pt(IV)Ac-POA may represent a new promising pharmacological agent in the treatment of malignant mesothelioma. Full article

Germline variants in the phosphatidylinositol glycan class A (PIGA) gene, which is involved in glycosylphosphatidylinositol (GPI) biosynthesis, cause multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) with X-linked recessive inheritance. The available literature has described a pattern of almost 100% X-chromosome inactivation in mothers carrying PIGA variants. Here, we report a male infant with MCAHS2 caused by a novel PIGA variant inherited from his mother, who has a non-skewed pattern of X inactivation. Phenotypic evidence supporting the pathogenicity of the variant was obtained by flow-cytometry tests. We propose that the assessment in neutrophils of the expression of GPI-anchored proteins (GPI-APs), especially CD16, should be considered in cases with variants of unknown significance with random X-inactivation in carrier mothers in order to clarify the pathogenic role of PIGA or other gene variants linked to the synthesis of GPI-APs. Full article

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus. Full article

Paslahepevirus balayani and Rocahepevirus ratti are genetically diverse species of hepatitis E virus [HEV]. Previously, only members of the Paslahepevirus genus were known to infect humans but recently some Rocahepevirus members have been found to be infectious to both immunocompromised and immunocompetent humans. Paslahepevirus balayani genotypes (gt) 1, 2, and 4 are known for their detrimental effects during pregnancy, causing pregnancy-related disorders. Recent findings have demonstrated the ability of Paslahepevirus balayani gt3 to replicate within placental cell lines, suggesting a direct effect on the placenta and fetus. To study whether zoonotic rat HEV strains possess a similar human-host placental tropism, we utilized JEG-3 cells to understand the replicative ability of an infectious clone of a recently reported strain of Rocahepevirus ratti, the LCK-3110 strain. Infectious cDNA clones of Pasla-, Avi-, and Rocahepevirus were transcribed and then, transduced into JEG-3 cells. Cells were harvested, and cell lysates were used for testing infectivity. Five days post-transfection or after inoculation onto naive HepG2/C3A cells, the cells were analyzed for infection. Replication in transduced JEG-3 cells and the infection potential in HepG2/C3A cells were assessed via an indirect immunofluorescence assay and a flow-cytometry assay. We found that the Rocahepevirus ratti LCK-3110 strain did not have efficient replication in JEG-3 cell cultures. Full article

Background: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. Methods: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. Results: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. Conclusion: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern. Full article