Flow-cytometry Assessment of DNA content and Immunophenotyping of Immune-cells in Lymph-node-specimens as a Potential Diagnostic Signature of Aggressiveness in B-Non-Hodgkin Lymphomas.

Flow-cytometry (FC) is a powerful tool that can assist in lymphoma diagnosis in lymph node (LN) specimens. Although lymphoma diagnosis and classification are mainly based on tumor cell characteristics, surrounding cells are less employed in this process. We retrospectively investigated alterations in the ploidy status, proliferative cell fraction (PF) and the percentages of surrounding immune cells in 62 consecutive LN specimens with B-Cell Non-Hodgkin Lymphoma (B-NHL) that were submitted for FC evaluation between 2019-2022. Compared with indolent B-NHLs, aggressive B-NHLs show increased DNA aneuploidy and PF, increased monocytes, immature-granulocytes, mature granulocytes, CD8+ T-cells, Double-Negative-T-cells and Double-Positive-T-cells, and decreased total CD45+ cells, total lymphocytes, CD4+ T-cells and CD4/CD8 ratio. Receiver operating characteristic analysis determined PF > 6.8% and immature-granulocytes > 0.9% as optimal cutoffs with highest specificity and sensitivity in differentiating aggressive and indolent B-NHLs. These findings further strength the diagnostic value of DNA content analysis by FC and suggest the utilization of tumor surrounding immune cells in NHL diagnosis and classification.

Conflicts of Interest in the Assessment of Chemicals, Waste, and Pollution.

Pollution by chemicals and waste impacts human and ecosystem health on regional, national, and global scales, resulting, together with climate change and biodiversity loss, in a triple planetary crisis. Consequently, in 2022, countries agreed to establish an intergovernmental science-policy panel (SPP) on chemicals, waste, and pollution prevention, complementary to the existing intergovernmental science-policy bodies on climate change and biodiversity. To ensure the SPP's success, it is imperative to protect it from conflicts of interest (COI). Here, we (i) define and review the implications of COI, and its relevance for the management of chemicals, waste, and pollution; (ii) summarize established tactics to manufacture doubt in favor of vested interests, i.e., to counter scientific evidence and/or to promote misleading narratives favorable to financial interests; and (iii) illustrate these with selected examples. This analysis leads to a review of arguments for and against chemical industry representation in the SPP's work. We further (iv) rebut an assertion voiced by some that the chemical industry should be directly involved in the panel's work because it possesses data on chemicals essential for the panel's activities. Finally, (v) we present steps that should be taken to prevent the detrimental impacts of COI in the work of the SPP. In particular, we propose to include an independent auditor's role in the SPP to ensure that participation and processes follow clear COI rules. Among others, the auditor should evaluate the content of the assessments produced to ensure unbiased representation of information that underpins the SPP's activities.

Laboratory Indices in Patients with Positive and Borderline Flow Cytometry Eosin-5-Maleimide-Screening Test Results for Hereditary Spherocytosis.

OBJECTIVE: To evaluate laboratory indices in patients with hereditary spherocytosis, with positive and borderline flow cytometry eosin-5-melamide (EMA)-bound red blood cells screening test. STUDY DESIGN: We compared laboratory indices of 151 samples obtained from 139 different individual patients with negative, borderline, or positive EMA-test results. We also compared the clinical data of the patients in each EMA test results group. RESULTS: Borderline EMA-test results were obtained for 13 patients and were associated with more severe anemia, and lower reticulocyte count and reticulocyte production index compared with samples with positive EMA-test results. A receiving operator characteristic analysis identified mean corpuscular hemoglobin concentration of <32.5 g/dL as a cut-off, between positive/borderline and negative test results with 100% sensitivity. A higher prevalence of clinical markers typical of hereditary spherocytosis was found in patients with borderline or positive compared with negative EMA test samples. CONCLUSIONS: Based on laboratory data, borderline EMA-test results may be an indication of a more severe form of hereditary spherocytosis. Using mean corpuscular hemoglobin concentration as a cut-off may help predict and reduce negative EMA tests without compromising sensitivity. This finding needs to be further validated in other flow cytometry laboratories with a large EMA test sample pool.

Brain-Derived Neurotrophic Factor (BDNF) Is Associated with Platelet Activity and Bleeding Tendency in Patients with Gaucher Disease.

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbß3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.

Clearance of the SARS-CoV-2 Virus in an Immunocompromised Patient Mediated by Convalescent Plasma without B-Cell Recovery.

Coronavirus disease (COVID-19) is a contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This case report presents a patient who had difficulty eradicating the corona virus due to being treated with Rituximab, which depletes B lymphocyte cells and therefore disables the production of neutralizing antibodies. The combined use of external anti-viral agents like convalescent plasma, IVIG and Remdesivir successfully helped the patient's immune system to eradicate the virus without B-cell population recovery. In vitro studies showed that convalescent plasma is the main agent that helped in eradicating the virus.

BDNF Val66Met is Associated with Pre-existing but not with Paclitaxel-induced Peripheral Neuropathy in an Israeli Cohort of Breast Cancer Patients.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that is important for the development, maintenance, and repair of the peripheral nervous system. The BDNF gene commonly carries a single nucleotide polymorphism (Val66Met-SNP), which affects the cellular distribution and activity-dependent secretion of BDNF in neuronal cells. OBJECTIVES: To check the association between BDNF Val66Met-SNP as a predisposition that enhances the development of chemotherapy-induced peripheral neuropathy in an Israeli cohort of patients with breast cancer who were treated with paclitaxel. METHODS: Peripheral neuropathy symptoms were assessed and graded at baseline, before beginning treatment, and during the treatment protocol in 35 patients, using the reduced version of the Total Neuropathy Score (TNSr). Allelic discrimination of BDNF polymorphism was determined in the patients' peripheral blood by established polymerase chain reaction and Sanger sequencing. RESULTS: We found Val/Val in 20 patients (57.14%), Val/Met in 15 patients (42.86%), and Met/Met in none of the patients (0%). Baseline TNSr scores were higher in Met-BDNF patients compared to Val-BDNF patients. The maximal TNSr scores that developed during the follow-up in Met-BDNF patients were higher than in Val-BDNF patients. However, exclusion of patients with pre-existing peripheral neuropathy from the analysis resulted in equivalent maximal TNSr scores in Met-BDNF and Val-BDNF patients. CONCLUSIONS: These observations suggest that BDNF Val66met-SNP has no detectable effect on the peripheral neuropathy that is induced by paclitaxel. The significance of BDNF Val66Met-SNP in pre-existing peripheral neuropathy-related conditions, such as diabetes, should be further investigated.

Brain-derived neurotrophic factor in hematological malignancies: From detrimental to potentially beneficial.

Emerging studies have highlighted brain-derived neurotrophic factor (BDNF), a neuronal growth factor abundant in the peripheral blood, and its tyrosine kinase receptor TRKB, as onco-genes and proteins that support the survival of malignant hematological cells. In contrast, other researchers reported on a favorable association between BDNF blood levels and prognosis, chemotherapy response and neurological side effects in patients with hematological malignancies. Here, we review the accumulated data regarding the expression of BDNF and its receptors in normal hematopoietic and lymphatic cells and tissue. In addition, in-vitro experiments, animal models and human sample studies that investigated the role of BDNF and its receptors in hematological malignancies are discussed. Finally, directions for future research aimed at revealing the mechanisms underlying the protective effect of BDNF in patients with these diseases are suggested.

Thymic involution, a co-morbidity factor in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating disease, characterized by extremely rapid loss of motor neurons. Our studies over the last decade have established CD4(+) T cells as important players in central nervous system maintenance and repair. Those results, together with recent findings that CD4(+) T cells play a protective role in mouse models of ALS, led us to the current hypothesis that in ALS, a rapid T-cell malfunction may develop in parallel to the motor neuron dysfunction. Here, we tested this hypothesis by assessing thymic function, which serves as a measure of peripheral T-cell availability, in an animal model of ALS (mSOD1 [superoxide dismutase] mice; G93A) and in human patients. We found a significant reduction in thymic progenitor-cell content, and abnormal thymic histology in 3-4-month-old mSOD1 mice. In ALS patients, we found a decline in thymic output, manifested in the reduction in blood levels of T-cell receptor rearrangement excision circles, a non-invasive measure of thymic function, and demonstrated a restricted T-cell repertoire. The morbidity of the peripheral immune cells was also manifested in the increase of pro-apoptotic BAX/BCXL2 expression ratio in peripheral blood mononuclear cells (PBMCs) of these patients. In addition, gene expression screening in the same PBMCs, revealed in the ALS patients a reduction in key genes known to be associated with T-cell activity, including: CD80, CD86, IFNG and IL18. In light of the reported beneficial role of T cells in animal models of ALS, the present observation of thymic dysfunction, both in human patients and in an animal model, might be a co-pathological factor in ALS, regardless of the disease aetiology. These findings may lead to the development of novel therapeutic approaches directed at overcoming the thymic defect and T-cell deficiency.

Low brain-derived neurotrophic factor protein levels and single-nucleotide polymorphism Val66Met are associated with peripheral neuropathy in type II diabetic patients.

BACKGROUND: Studies by our group demonstrated brain-derived neurotrophic factor (BDNF) levels in blood and BDNF-Val66met-SNP as potential biomarkers in chemotherapy-induced peripheral neuropathy. Here, we evaluate symptoms of peripheral neuropathy (PN) and depression in patients with type II diabetes mellitus in search of an association with serum BDNF levels and the Val66Met-SNP. METHODS: In total, 90 patients enrolled in the study; 23 (25.6%) had known PN, as determined by nerve conduction studies (NCS-PN), and 67 (74.4%) were not diagnosed with PN (U-PN). PN symptoms were assessed and graded in these groups using the total neuropathy score (TNSr) and DN4 scales. Small nerve fiber testing of sensitivity thresholds to cold, warm and hot pain signals was performed using the Q-sense device. Depression was assessed using the PHQ9 questionnaire. BDNF protein levels and Val66Met-SNP were determined with ELISA and Sanger sequencing, respectively. RESULTS: NCS-PN patients showed lower serum BDNF levels alongside significantly higher TNSr, DN4 and PHQ9 scores and lower hot pain sensitivity thresholds as compared to U-PN patients. Patients with Met-BDNF-SNP showed increased TNSr scores and lower hot pain sensitivity thresholds as compared to patients with Val-BDNF-SNP. Depression showed a weaker correlation with sensitivity thresholds to hot pain signals as compared to TNSr and DN4 scores. CONCLUSIONS: Diminished peripheral BDNF resources and Met-BDNF-SNP genotype are associated with augmented symptoms of PN in patients with type II diabetes mellitus. Sensitivity thresholds to hot pain signals may be less influenced by depression and possibly more accurately detect PN symptoms in diabetic patients.

Flow cytometry aneuploidy and cell cycle indexing as a possible tool for differentiating between CD10+ diffuse large B-cell lymphoma and follicular lymphoma.

BACKGROUND: Differential diagnosis between diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) becomes a challenge when adequate biopsy is unavailable. The present study aimed to investigate the applicability of DNA cell cycle analysis by flow cytometry (FC) for differentiating between CD10+ DLBCL and FL. METHODS: Data were collected from 57 specimens where CD5- /CD10+ /light chain restricted B cells were detected. DNA staining was performed using the Coulter DNA Prep Kit. Cell cycle fractions were evaluated by automatic analysis using the ModFit LT software. RESULTS: Histopathological analysis confirmed the diagnosis of CD10+ FL in 30 specimens (52.6%), CD10+ DLBCL in 24 specimens (42.1%), and CD10+ Burkitt lymphoma in 3 specimens (5.3%). A significantly higher rate of DNA aneuploidy was detected among CD10+ DLBCL than FL specimens (50 vs. 13.3% respectively, p = .003). Likewise, DNA index was significantly higher in CD10+ DLBCL relative to FL (1.26 ± 0.35 vs. 1.04 ± 0.16 respectively, p = .004). Notably, the proportion of cells in the S-phase and proliferative fraction was significantly higher in CD10+ DLBCL than in CD10+ FL (S-phase: 15.97 ± 13.94 vs. 4.43 ± 4.41 mean ± SD, respectively, p < .0001; proliferative fraction: 18.87 ± 15.17 vs. 5.78 ± 7.04 mean ± SD, respectively, p = .0001). Using a receiver operating characteristic analysis, optimal cutoffs for S-phase ≥7% and proliferative fraction ≥9% were determined. These values could be used to differentiate between CD10+ DLBCL and CD10+ FL. CONCLUSION: Including DNA cell cycle analysis in the FC lymphoma assessment panel may be of diagnostic value in differentiating between CD10+ DLBCL and FL when adequate biopsy is unavailable.

Attenuation of AD-like neuropathology by harnessing peripheral immune cells: local elevation of IL-10 and MMP-9.

Immunization with an altered myelin-derived peptide (MOG45D) improves recovery from acute CNS insults, partially via recruitment of monocyte-derived macrophages that locally display a regulatory activity. Here, we investigated the local alterations in the cellular and molecular immunological milieu associated with attenuation of Alzheimer's disease-like pathology following immunotherapy. We found that immunization of amyloid precursor protein/presenilin 1 double-transgenic mice with MOG45D peptide, loaded on dendritic cells, led to a substantial reduction of parenchymal and perivascular amyloid beta (Abeta)-plaque burden and soluble Abeta((1-42)) peptide levels as well as reduced astrogliosis and levels of a key glial scar protein (chondroitin sulphate proteoglycan). These changes were associated with a shift in the local innate immune response, manifested by increased Iba1+/CD45(high) macrophages that engulfed Abeta, reduced pro-inflammatory (tumor necrosis factor-alpha) and increased anti-inflammatory (interleukin-10) cytokines, as well as a significant increase in growth factors (IGF-1 and TGFbeta) in the brain. Furthermore, the levels of matrix metalloproteinase-9, an enzyme shown to degrade Abeta and is associated with glial scar formation, were significantly elevated in the brain following immunization. Altogether, these results indicate that boosting systemic immune cells leads to a local immunomodulation manifested by elevated levels of anti-inflammatory cytokines and metalloproteinases that contribute to ameliorating Alzheimer's disease pathology.

Prediction of Chemotherapy-Induced Peripheral Neuropathy in Patients with Lymphoma and Myeloma: the Roles of Brain-Derived Neurotropic Factor Protein Levels and A Gene Polymorphism.

BACKGROUND AND PURPOSE: Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that plays an essential role in the maintenance of the nervous system. We have evaluated the peripheral blood protein levels of BDNF and the valine-to-methionine substitution at codon 66 (Val66Met) single-nucleotide polymorphism (SNP) as potential biomarkers for the early recognition of chemotherapy-induced peripheral neuropathy (CIPN) in non-Hodgkin lymphoma and multiple myeloma patients. METHODS: CIPN was assessed in 45 patients at the diagnosis and during vincristine or bortezomib-based therapy using objective [reduced version of the Total Neuropathy Score (TNSr)] and subjective (FACT-GOG-NTx) tools. Depression was assessed using the Patient Health Questionnaire-9 (PHQ-9) questionnaire. BDNF protein levels and the Val66Met SNP were determined using ELISA and Sanger sequencing. RESULTS: The pretreatment BDNF protein level was inversely correlated with the maximum TNSr, FACT-GOG-NTx, and PHQ-9 scores in both genotypes. BDNF patients with the Val/Val genotype demonstrated significantly higher maximum FACT-GOG-NTx and PHQ-9 scores than those with the Val/Met and Met/Met genotypes (Met-BNDF carriers). Correlations between PHQ-9 and TNSr score were found only in Met-BDNF carriers, suggesting that peripheral neuropathy and depression coincide in Met-BDNF carriers. CONCLUSIONS: Determining the BDNF protein levels before initiating chemotherapy might be a useful tool for CIPN risk assessment and preemptive dose modification. The present data should be validated in larger studies that include other neurotoxic agents.

Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection.

Multiple sclerosis (MS) is characterized by lesions with inflammatory infiltration, demyelination and axonal damage in the CNS white matter that correlates with the extent of disease disability. Knowledge of up-regulatory triggers of neuroprotective pathways in the CNS is essential for the development of the next generation of disease therapies. Recent studies have suggested a neuroprotective activity of the lesion-infiltrating immune cells. We studied the secretion of brain-derived neurotrophic factor (BDNF) from the immune cells of untreated patients with relapsing remitting (RR) MS with mild to moderate disability and sought immune factors that regulate the BDNF levels and affect the survival of neuronal cells in vitro. We found lower than normal secreted levels of BDNF from the immune cells of these patients. The normal effect of CD40 stimulation that up-regulates BDNF secretion levels and induces neuroprotection was absent in the MS patients, while the expression of CD40 on their monocytes was elevated. The failure of BDNF availability from immune cells in patients with RR-MS and the loss of a neuroprotective effect by these cells may be related to a more widespread phenomenon of deviated immunity in MS, and may be linked to the continuous CNS neuronal tissue loss during the course of this disease.

Elevated serum BDNF levels are associated with favorable outcome in CLL patients: Possible link to CXCR4 downregulation.

Increased chemokine C-X-C receptor 4 (CXCR4) expression is related to unfavorable outcome in chronic lymphocytic leukemia (CLL). Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that has been shown previously to interact with CXCR4 in neuronal cells. Here, we studied the in vitro effect of BDNF on CXCR4 expression and chemotaxis toward stromal derived factor-1 (SDF-1) in freshly isolated CLL cells. We also explored the correlations between serum BDNF levels in CLL patients and disease characteristics and clinical course. Incubation of CLL cells with recombinant BDNF (50 ng/mL) resulted in a downregulation of CXCR4 surface expression and atenuated chemotaxis toward SDF-1. Higher serum BDNF levels were associated with a mutated immunoglobulin heavy chain variable (IGHV) gene, an early clinical stage, and a stable clinical course. Our findings suggest that increased circulating blood BDNF may be associated with a favorable effect in CLL. However, the exact mechanism of this favorable effect should be investigated further.