Peripheral Blood Granulocytes Research Articles

The Effect of Orally Administered Multi-Strain Probiotic Formulation (Lactobacillus, Bifidobacterium) on the Phagocytic Activity and Oxidative Metabolism of Peripheral Blood Granulocytes and Monocytes in Lambs.

Probiotic feed additives have attracted considerable research interest in recent years because the effectiveness of probiotics can differ across microbial strains and the supplemented macroorganisms. The present study was conducted on 16 lambs divided equally into two groups (C-control and E-experimental). The examined lambs were aged 11 days at the beginning of the experiment and 40 days at the end of the experiment. The diet of group E lambs was supplemented with a multi-strain probiotic formulation (Lactobacillus plantarum AMT14, Lactobacillus plantarum AMT4, Lactobacillus rhamnosus AMT15, and Bifidobacterium animalis AMT30), whereas group C lambs did not receive the probiotic additive. At the beginning of the experiment (day 0) and on experimental days 15 and 30, blood was sampled from the jugular vein to determine and compare: phagocytic activity (Phagotest) and oxidative metabolism (Phagoburst) of peripheral blood granulocytes and monocytes by flow cytometry. An analysis of the phagocytic activity of granulocytes and monocytes revealed significantly higher levels of phagocytic activity (expressed as the percentage of phagocytic cells and mean fluorescence intensity) in lambs that were administered the multi-strain probiotic formulation compared with lambs in the control group. The probiotic feed additive also exerted a positive effect on the oxidative metabolism of both granulocytes and monocytes (expressed as the percentage of oxidative metabolism and mean fluorescence intensity) after stimulation with Escherichia coli bacteria and with PMA (4-phorbol-12-β-myristate-13-acetate). These findings suggest that the tested probiotic formulation may have a positive effect on the immune status of lambs.

Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel.

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

Modern management of leukemoid reactions

ABSTRACT. Leukemoid reaction (LR) is a transient hematological syndrome of reactive nature characterized by persistent leukocytosis of more than 50,000 cells/µL. LR can be caused by a variety of factors, including infections, intoxications, malignant neoplasms, severe hemorrhages, or acute hemolysis.
 The most common form of LR is neutrophilic myeloid reaction, which occurs in 80-90 % of cases. The diagnosis of LR is based on clinical examination and laboratory test results, including complete blood count with differential leukocyte formula, determination of leukocyte alkaline phosphatase level, aspiration and biopsy of bone marrow.
 Differential diagnosis of LR from other forms of leukemia is a challenging task. For this, it is necessary to conduct a thorough examination, including cytogenetic and molecular analysis of peripheral blood and bone marrow granulocytes. Despite certain advances in the differential diagnosis of LR, the mortality rate of patients in the world remains high, which requires the attention of doctors of various profiles.
 Treatment of LR depends on the underlying cause that caused it. The article proposes a diagnostic and treatment algorithm for LR in the case of its toxico-infectious origin.

Density Gradient Centrifugation-Independent Purification of Human Basophils.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.

Ex Vivo 13C-Metabolic Flux Analysis of Porcine Circulating Immune Cells Reveals Cell Type-Specific Metabolic Patterns and Sex Differences in the Pentose Phosphate Pathway.

In general, females present with stronger immune responses than males, but scarce data are available on sex-specific differences in immunometabolism. In this study, we characterized porcine peripheral blood mononuclear cell (PBMC) and granulocyte energy metabolism using a Bayesian 13C-metabolic flux analysis, which allowed precise determination of the glycolytic, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) fluxes, together with an assessment of the superoxide anion radical (O2•-) production and mitochondrial O2 consumption. A principal component analysis allowed for identifying the cell type-specific patterns of metabolic plasticity. PBMCs displayed higher TCA cycle activity, especially glutamine-derived aspartate biosynthesis, which was directly related to mitochondrial respiratory activity and inversely related to O2•- production. In contrast, the granulocytes mainly utilized glucose via glycolysis, which was coupled to oxidative PPP utilization and O2•- production rates. The granulocytes of the males had higher oxidative PPP fluxes compared to the females, while the PBMCs of the females displayed higher non-oxidative PPP fluxes compared to the males associated with the T helper cell (CD3+CD4+) subpopulation of PBMCs. The observed sex-specific differences were not directly attributable to sex steroid plasma levels, but we detected an inverse correlation between testosterone and aldosterone plasma levels and showed that aldosterone levels were related with non-oxidative PPP fluxes of both cell types.

Prediction of the risk of recurrence of the leprosy process

The aim of the study was to study the degree of neutrophil granulocyte myeloperoxidase activity in peripheral blood of patients with leprosy in order to predict the possible recurrence of the infectious process.Materials and methods. Electron grams of biopsies of the affected skin and blood smears of 8 (5 women and 3 men) patients with borderline (BB), 9 (6 women and 3 men) patients with subpolar lepromatous (LLs) and 7 (4 women and 3 men) patients with polar lepromatous (LLr) were used as the object of the study forms of leprosy. At the ultramicroscopic level, the degree of MP activity detected by the electron-cytochemical method in the Mf of biopsies of the affected skin, both first registered before the start of therapy and with the activation of the process during treatment, was taken into account. The percentage of mitochondria with MP Mf activity relative to the MP of inactive cells was calculated. In peripheral blood smears, the activity of neutrophilic granulocytes (NG) MP was evaluated by the mean cytochemical coefficient (SCC).Results. It was found that in patients with the highest percentage of peroxidase-active mitochondria (79-92%), the presence of MP activity on the membranes of phagosomes and in the electron-transparent zone (around M. leprae) correlated with rapid and persistent regression of the leprosy process and long periods of relapse-free period, which was determined by comparing the data of medical histories with the results obtained in during our research. It was found that in patients without relapse with forms of BB and LLs leprosy, the activity of MP in neutrophils was 1.3 times higher (p<0.05), and the percentage of peroxidase-active mitochondria was 3 times higher (p<0.01) compared with these indicators of patients with relapse of the disease. When comparing the studied markers of groups of patients without relapse with the form of leprosy LLp, the degree of MP NG activity increased by 1.4 times (p<0.05), the percentage of mitochondria with MP Mf activity increased by 3 times (p<0.01) relative to patients with relapses.Conclusion. Thus, the determination of myeloperoxidase activity of neutrophil granulocytes of peripheral blood can act as a prognostic marker for determining the effectiveness of anti-leprosy therapy and the risk of relapses of the disease during and after treatment.

Combinatorial BCL-2/ BCL2L1 Expression Predicts Clinical Response to Ruxolitinib in Myelofibrosis

Introduction: JAK inhibitors (JAKi) marked a revolutionary breakthrough in the treatment of patients (pts) with myelofibrosis (MF). The BCL-2 family of proteins have been implicated in both disease development and mechanisms of response/resistance to JAKi. This is further reinforced by preliminary evidence of therapeutic efficacy of combining JAK2 and BCL-xL inhibition. Aims&Methods: In the current study, we investigated the expression of three major BCL-2 family members in MF pts treated with ruxolitinib (RUX) at baseline and during treatment. Gene expression of BCL-2, BCL2L1 (encoding BCL-xL) and MCL-1 was analyzed by qPCR normalized to endogenous 18S gene using cDNA obtained from peripheral blood (PB) granulocytes; the relative gene expression (expressed as fold-change, FC) was calculated with reference to healthy controls using the Ct (2−ΔΔCt) method. Results: The study included 19 pts (10 males; median age 61 years) with MF (10 primary, 9 secondary) who received treatment with RUX. Sixteen pts were JAK2-mutated, 2 CALR-mutated, and 1 MPL-mutated. Treatment response was fully evaluable across the entire treatment period. After a median treatment time of 67 (range, 6-121) months, 7 (37%) pts achieved a spleen response (defined as a >50% reduction in palpable splenic length lasting >6 months) and were considered as responders, as compared to 12 (63%) non-responders. We did not find any significant correlation of baseline BCL-2/ BCL2L1/ MCL-1 expression with clinical and molecular characteristics, the only exception being the association between EZH2 mutation and higher BCL2L1 expression. First, we ascertained whether BCL2 family protein expression levels at baseline predicted response to RUX. We found that BCL2 and BCL2L1 were significantly more expressed in responders compared to non-responders, with median FC values of 0.14 vs 0.04 ( P=.0130) and 2.46 vs 0.51 ( P=.0096), respectively (Fig. 1A). A similar, not statistically significant, trend was observed for MCL-1 expression. Accordingly, in univariate regression logistic analysis, both BCL2 and BCL2L1 FCs correlated significantly with the probability of response to RUX. Then, we incorporated BCL-2 and BCL2L1 gene expression at baseline into a combinatorial score (CS) resulting from the product of the respective FCs (FC BCL-2 * FC BCL-xL). The CS outperformed the individual BCL2 family proteins expression in predicting RUX response, as confirmed in regression logistic analysis. ROC analysis identified 0.06 as the optimal cut-off value of the CS. In univariate analysis, pts with a baseline CS >0.06 had a significantly higher probability of responding to RUX (OR 27.5, 95% CI 2.1-236.7; P=.0037), with a superior cumulative incidence of RUX response at 24 weeks (50% vs 9%, respectively; P=.0045) (Fig. 1B). Finally, we prospectively assessed the changes in gene expression during treatment with RUX. Six out of seven responder pts had an available PB sample at the time of best response, while all non-responders were evaluated at some point during RUX therapy. Among responders, 5 lost response after a median time of 34 months, and 3/5 had an available sample for analyses at the time of response loss. Although we did not find any significant difference comparing gene expression at baseline and on treatment, we observed a trend for BCL-2 upregulation in responders at the time of best response. Furthermore, non-responders showed a down-regulation of MCL-1 expression during treatment ( P=.05). By comparing gene expression of responder pts at the time of best vs loss of response, we discovered a down-regulation for all the three genes, with a statistical significance that was reached only for BCL-2 ( P=.0471). Conclusion: Despite study limitations (mainly the small cohort), this preliminary data suggests that the expression levels of BCL2 family proteins may represent a predictor of response to RUX. Specifically, we developed a simple, combinatorial score includingbaseline BCL-2 and BCL2L1 expression that accurately discriminated pts who responded to RUX. Moreover, we showed that BCL2 family protein expression changes during RUX treatment, with potential correlations with both response achievement and loss. Further studies are needed in order to confirm and expand our data.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

Phase I Trial of Allogeneic Donor Gamma Delta T Cell Infusion Post-Hematopoietic Cell Transplantation, an Interim Safety Report

Background Patients with 2017 European LeukemiaNet (ELN) adverse risk acute myeloid leukemia (AML) are at 40% risk of relapse leading to poor survival after allogeneic hematopoietic cell transplantation (allo-HCT) (Hansen TCT 2021, Jimenez BMT 2021). Ex vivo donor-derived gamma delta T cells (GDT) expanded with artificial antigen presented calls (aAPC) are a novel therapy with potent MHC-independent antineoplastic cytotoxicity. An ongoing phase 1/1b study is evaluating the safety and potential efficacy of ex vivo expanded donor GDT after reduced-intensity conditioning (RIC) allo-HCT in patients with ELN adverse risk AML ( NCT05015426). We report on manufacturing, safety, and correlative biology of expanded donor GDT after allo-HCT in the first 5 treated patients (Table 1). Methods The primary endpoint is the maximum-tolerated dose (MTD) of allogeneic donor GDT as a single infusion at day 60 after RIC allo-HCT. Next generation sequencing was used for measurable residual disease (MRD) assessment pre-HCT and post-GDT infusion. Donor GDT collected from the same donor of the allo-HCT were enriched from non-mobilized leukapheresis product by stimulation with zoledronic acid and IL-2 for 7 days followed by alpha beta T cell depletion using the Miltenyi CliniMACS. Enriched GDT (>90% TCRVδ2) were then co-cultured with irradiated aAPCs in Wilson Wolf G-Rex 100MCS over 10 days (Boucher J. Immunother 2023). This single center, investigator-initiated phase 1 trial is testing 3 distinct dose cohorts of infused GDT (5 x 10 6 cells/kg, 2.5 x 10 7 cells/kg, and 1 x 10 8 cells/kg) with acceptable range of 25% margin. A Bayesian optimal interval design is used to guide dose escalation/de-escalation decisions. Results First 3 patients enrolled in dose level 1 (DL1) cohort received GDT dose of 6.25 x 10 6 cells/kg, while last 2 patients enrolled in DL2 cohort received 3.13 x 10 7 cells/kg dose. Dose-limiting toxicities (DLT) were evaluated for 42 days following GDT infusion and no DLT were observed. None of the treated patients experienced cytokine release syndrome (CRS), Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) or graft-versus-host disease (GVHD) during DLT period. Among patients treated on DL1, patient #01 had ASXL1 AML with MRD pos (VAF 43.9%) prior to RIC haploidentical (Haplo) HCT. He is in ongoing complete remission (CR) 9 months post-HCT and has had no GVHD. Patient #02 had MRD pos (VAF 61.7%) AML with TP53 mutation/complex karyotype prior to RIC matched sibling donor allo-HCT. He is in CR and has mild chronic GVHD at 7 months post-HCT. Patient #03 with pre-HCT MRD negRUNX1 AML is in ongoing CR 7 months post-RIC Haplo HCT with no GVHD. At day 28 and day 120 post-GDT, all 3 patients were MRD neg in bone marrow and had 100% bone marrow and peripheral blood leukocyte and granulocyte chimerism. In DL2 cohort, patient #04 with RUNX1 AML and patient #05 with t(9;22) AML both were MRD neg prior to RIC Haplo HCT and remain in CR with no GVHD at 5 and 4 months post-HCT, respectively. At day 28 post-GDT, both patients were MRD neg and had 100% chimerism. Patient #05 had a fall during DLT period in a setting of orthostatic hypotension requiring hospitalization for 24 hours for hydration. This was the only grade 3 adverse event observed in this trial. Serum cytokines analyzed included IL2, IL6, IL15, IFNγ, TNFα, Angiopoietin 1&2, and GM-CSF (Figure 1). We compared results prior and up to 90 days post-GDT infusion and found no significant differences, which can explain no CRS or ICANS events yet observed on this trial. Immunophenotype (TCRVδ2, TCRVδ1, PD1, CTLA4, TIGIT and CD28) of GDT was analyzed in samples of donor apheresis, pre- and post-enrichment, infused GDT product and at days 28 and 90 post-infusion. GDT (TCRVδ2) persisted at day 28 and day 90 post-infusion and expressed low levels of cell surface inhibitory receptors. Conclusion This preliminary interim report demonstrates favorable safety and tolerability of donor GDT therapy with no CRS, ICANS or GVHD in first 5 patients treated in this trial. All these patients with adverse risk AML are in ongoing MRD neg CR following RIC allo-HCT, including 2 pre-HCT MRD pos cases with TP53 AML and AXSL1 AML. Early results of this trial are promising, which provides validation for continued testing of adoptive transfer of ex vivo expanded donor GDT. Updated results of this trial will be presented at the annual conference.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

KRT17high/CXCL8+ Tumor Cells Display Both Classical and Basal Features and Regulate Myeloid Infiltration in the Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is generally divided in two subtypes, classical and basal. Recently, single-cell RNA sequencing has uncovered the coexistence of basal and classical cancer cells, as well as intermediary cancer cells, in individual tumors. The latter remains poorly understood; here, we sought to characterize them using a multimodal approach. We performed subtyping on a single-cell RNA sequencing dataset containing 18 human PDAC samples to identify multiple intermediary subtypes. We generated patient-derived PDAC organoids for functional studies. We compared single-cell profiling of matched blood and tumor samples to measure changes in the local and systemic immune microenvironment. We then leveraged longitudinally patient-matched blood to follow individual patients over the course of chemotherapy. We identified a cluster of KRT17-high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines. The proportion of KRT17high/CXCL8+ cells in patient tumors correlated with intratumoral myeloid abundance, and, interestingly, high protumor peripheral blood granulocytes, implicating local and systemic roles. Patient-derived organoids maintained KRT17high/CXCL8+ cells and induced myeloid cell migration in a CXCL8-dependent manner. In our longitudinal studies, plasma CXCL8 decreased following chemotherapy in responsive patients, while CXCL8 persistence portended worse prognosis. Through single-cell analysis of PDAC samples, we identified KRT17high/CXCL8+ cancer cells as an intermediary subtype, marked by a unique cytokine profile and capable of influencing myeloid cells in the tumor microenvironment and systemically. The abundance of this cell population should be considered for patient stratification in precision immunotherapy. See related commentary by Faraoni and McAllister, p. 2297.

Preliminary Comparison of Molecular Antioxidant and Inflammatory Mechanisms Determined in the Peripheral Blood Granulocytes of COVID-19 Patients.

The aim of this study was to evaluate selected parameters of redox signaling and inflammation in the granulocytes of COVID-19 patients who recovered and those who died. Upon admission, the patients did not differ in terms of any relevant clinical parameter apart from the percentage of granulocytes, which was 6% higher on average in those patients who died. Granulocytes were isolated from the blood of 15 healthy people and survivors and 15 patients who died within a week, and who were selected post hoc for analysis according to their matching gender and age. They differed only in the lethal outcome, which could not be predicted upon arrival at the hospital. The proteins level (respective ELISA), antioxidant activity (spectrophotometry), and lipid mediators (UPUPLC-MS) were measured in the peripheral blood granulocytes obtained via gradient centrifugation. The levels of Nrf2, HO-1, NFκB, and IL-6 were higher in the granulocytes of COVID-19 patients who died within a week, while the activity of cytoplasmic Cu,Zn-SOD and mitochondrial Mn-SOD and IL-2/IL-10 were lower in comparison to the levels observed in survivors. Furthermore, in the granulocytes of those patients who died, an increase in pro-inflammatory eicosanoids (PGE2 and TXB2), together with elevated cannabinoid receptors 1 and 2 (associated with a decrease in the anti-inflammatory 15d-PGJ2), were found. Hence, this study suggests that by triggering transcription factors, granulocytes activate inflammatory and redox signaling, leading to the production of pro-inflammatory eicosanoids while reducing cellular antioxidant capacity through SOD, thus expressing an altered response to COVID-19, which may result in the onset of systemic oxidative stress, ARDS, and the death of the patient.

Association Between SPARC Polymorphisms and Ankylosing Spondylitis and Its mRNA and Protein Expression in a Chinese Han Population: A Case-Control Study.

We explore the association of polymorphisms in Secreted protein acidic and rich in cysteine (SPARC) with ankylosing spondylitis (AS) and detect SPARC mRNA and protein expression in a Chinese Han population. Nine single-nucleotide polymorphisms (SNPs) of SPARC were genotyped in 768 AS patients and 768 controls by TaqMan genotyping assay. mRNA expression of SPARC was detected by real-time polymerase chain reaction (RT-PCR), and serum level of SPARC protein was detected by ELISA. The frequency of A allele of rs171121187 was significantly higher in AS patients than in controls (Pc=0.003, odds ratio [OR]=1.45, 95% confidence interval [95% CI] = 1.18-1.77), the AA and AC genotypes increased the risk of AS when compared with CC genotype (Pc=0.003, OR=3.96, 95% CI=1.80-8.75, and Pc=0.003, OR=1.27, 95% CI=1.01-1.61, respectively). The frequency of G allele of rs4958487 was significantly lower in AS than in controls (Pc=0.001, OR=0.60, 95% CI=0.47-0.68), the GG and GA genotypes reduced the risk of AS when compared with AA genotype (Pc=0.005, OR=0.46, 95% CI 0.18-1.14, and Pc=0.005, OR=0.60, 95% CI=0.45-0.79, respectively). The haplotype AA of rs17112187/rs4958487 significantly increased the risk of AS (P=2.31E-5, OR=1.60, 95% CI=1.28-1.98), while haplotype CG decreased the risk of AS (P=5.42E-5, OR=0.55, 95% CI=0.41-0.74). Expression levels of SPARC mRNA were significantly lower in both Peripheral blood mononuclear cells (PBMC) and granulocytes in AS patients than in controls (P=0.008 and P=0.005, respectively). SPARC protein levels were also reduced in AS patients versus the controls (P=0.002). This study indicates that polymorphisms in SPARC are associated with AS susceptibility, and both mRNA and protein levels of SPARC are decreased in AS patients in a Chinese Han population.

XISTexpression in male leukocytes

Abstract XISTexpression occurs in human somatic cells possessing more than one X chromosome to balance gene expression and in male germline cells to ensure X chromosome inactivation. A study designed to investigate the sex-biased expression of X-linked immune genes convincingly and repeatedly found low-level XISTexpression in male leukocytes. RNA was extracted from peripheral blood mononuclear cells (PBMC), granulocytes, and oral mucosa from healthy young adults of both sexes (n = 8) by the TRIzol method. After a DNase treatment, RNA was reverse transcribed with a mix of random hexamers and oligodT primers. Real-time PCR was performed in duplicate on 5 ng RNA-equivalent cDNA in duplicate using Taqman primer-probe sets. Negative controls included non-template controls and non-reverse transcribed samples; ref gene, ACTB.Delta Ct between parametric groups were compared by Welch’s t-test. Control genes behaved as expected: CDH1, was expressed higher in mucosa than in leukocytes (P < 0.04); the Y-linked gene TSMB4Ywas expressed in male cells only. Most X-linked immune genes were expressed higher in leukocytes than in mucosa (exception TLR7) with an equal expression between the sexes (0.3< P>0.93) or a sex-biased expression: FOXP3(male-biased, P= 0.04), SH2D1A (female-biased, P= 0.003). Only XISTexpression differed from the expected profile, as it was also found in all male leukocyte samples, both PBMC and granulocytes, but not in mucosa samples. The expression levels in male leukocytes were much lower than in female leukocytes (P< 0.0001). Further investigation on XISTexpression in male leukocytes will focus on chromosomal localization of lncRNA XIST within male nuclei and the life cycle stage of XIST-expressing male leukocytes. Supported by UDEM grant UIN20512

Hematological features of patients with type 2 diabetes depending on the variant of SARS-COV-2

The aim of our work was to investigate the peculiarities of hematological indicators in patients with COVID-19 depending on the variant of SARS-CoV-2. A retrospective study of the electronic medical records of 19 patients with the delta variant (7 patients had comorbidity with type 2 diabetes (T2D); 12 patients without T2D - control group) and 46 patients with the omicron variant (26 patients had comorbidity with T2D; 20 patients without T2D - control group). No statistically significant differences were found in gender, the number of leukocytes, lymphocytes, and granulocytes in peripheral blood, C-reactive protein, and D-dimer. The results also showed a significant difference in procalcitonin level and monocyte count in patients with delta variant and T2D. A predictive model was developed using binary logistic regression to determine the SARS-CoV-2 variant based on hematological parameters (sensitivity - 76.5%, specificity - 84.8%). Thus, the peculiarities of hematological indicators of patients with T2D depending on the variant of SARS-CoV-2 (delta or omicron) were established.

The effect of sodium thiosulfate on immune cell metabolism during porcine hemorrhage and resuscitation.

Sodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers. After 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA). PBMCs showed significantly higher mitochondrial O2 uptake and lower production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis. In a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods.

Correlation of High Eosinophil Count Levels in Blood with Metastasis in Colorectal Carcinoma

Background: Eosinophils are bone marrow-derived granulocytes in peripheral blood and tissues primarily involved as effector cells in colorectal cancer metastasis. High levels of eosinophils correlate with poor prognosis in colorectal cancer. Aim: To prove the correlation between increased levels of eosinophils with clinical stage according to the TNM system and metastatic status in patients with colorectal carcinoma. Methods: An analytical cross-sectional study was conducted from July 2020 to June 2021 at Prof. Dr. IGNG Ngoerah Hospital. Data analysis in this study consisted of univariate analysis, bivariate analysis, and multivariate analysis using SPSS IBM version 23. Results: Obtained 40 subjects patient colorectal cancer where men were 25 (62.5%), mean age ± SD53.15 ± 12.5 years, normal nutritional status 29 (72.5%), most locations in the rectum 16 (40%), followed by descending colon 15 (37.5%), the most clinical-stage IVA 13 (32.5%), lung metastases 12 (30%) with adenocarcinoma type 34 (90%). The average level of eosinophils±SDobtained445.63±283.03. The correlation between eosinophil levels and the clinical stage is strongly positive (r = 0.604; p < 0.001), and with the incidence of distant metastases, a strong positive correlation (r = 0.651; p<0.001). Conclusion: There is a statistically significant correlation between increased levels of eosinophils in the blood in metastases and stages colorectal cancer patients, where the higher the eosinophil count levels, the higher the stage and an increase in the incidence of metastases.

ICoreDrop: A robust immune monitoring spectral cytometry assay with six open channels for biomarker flexibility.

Recent advances in spectral cytometry have extended our ability to monitor immune cell subsets and activation status while simultaneously improving rare population detection. However, technical challenges in reference control selection and autofluorescence extraction serve as barriers to broad application of spectral flow cytometry. Furthermore, the complexity of spectral cytometry panel development limits the adaptation of established assays. Here, we describe the development of a spectral immunophenotyping assay with robust drop-in capability to enable biomarker interrogation flexibility. The immune monitoring core (iCore), which can be used in part or total, captures broad and granular immune subsets across T cells, B cells, NK cells, monocytes, dendritic cells, and granulocytes in peripheral whole blood. Additional user-selected biomarkers can be dropped in (Drop) using channels BV421, Alexa Fluor 488, PE, PE-Cy7, APC, and APC-Cy7. A comprehensive assessment of reagent and panel performance was conducted, including reference control comparison and optimal autofluorescence (AF) extraction on the 5-laser Cytek Aurora system for healthy donor blood. Assay precision and stability analyses revealed robust intra-assay precision, with 95% of 83 distinct population gates having <20% CV. In the presence of additional drop-in markers in two different settings, a T cell module and a myeloid/B cell module, the drop-in channels themselves achieved <20% CV across 12 out of 13 additionally queried population gates. Overall, establishment of optimal unmixing practices will enable widespread adoption of spectral cytometry assays.

Expression and function of receptors for the formylated peptides in granulocytes of the patients with rheumatoid arthritis

Formyl peptide receptors (FPRs) are an important part of innate immunity involved in antimicrobial phagocyte functions such as chemotaxis, secretory degranulation, and respiratory burst. These phagocyte responses are observed in both acute and systemic chronic inflammation. Abundant or constant release of pro-inflammatory ligands leads to the pre-activation of phagocytes when subsequent stimulation induces more intense cellular response. Binding of the formyl peptide receptor with its agonist activates production of reactive oxygen species, due to triggering phosphorylation of the cytoplasmic subunits p47phox and p67phox followed by their translocation to the plasma membrane and assembly into the NADPH oxidase complex. Rheumatoid arthritis is characterized by an imbalance of immune processes and autoimmune responses against the own joint tissues. It is known that, granulocytes produce increased amounts of oxygen radicals in various pathologies, including rheumatoid arthritis. We suggest that such enhancement may be due to increased expression of formyl peptide receptors or components of the FPR/PKC/NOX2 signaling pathway. Our aim was to study the mRNA expression of fpr1/fpr2 genes and the FPR-dependent production of reactive oxygen species by isolated peripheral blood granulocytes from the patients with rheumatoid arthritis. Materials and methods. The objects of the study were isolated peripheral blood granulocytes. We analyzed, respectively, 166 and 85 samples from the patients with rheumatoid arthritis and healthy donors. The production of reactive oxygen species was assessed using luminol-dependent chemiluminescence. For FPR1 activation we used a distinct concentration of the formyl peptide fMLF: the response to it was completely inhibited by pretreatment of the cells with FPR1 antagonist N-t-boc-MLF. FPR2 activation was performed by synthetic peptide WKYMVM, a specific receptor agonist. In the patients with rheumatoid arthritis, we have revealed an increased level of spontaneous and phorbol ester-induced production of reactive oxygen species by isolated peripheral blood granulocytes, thus reflecting a pre-activated state of the phagocytes in rheumatoid arthritis. We have found the increased FPR1-mediated production of oxygen radicals and expression of mRNA of fpr1 gene in blood granulocytes of rheumatoid arthritis patients. Furthermore, the enhancement of oxidase function may be associated with constitutive activation of the FPR1/PKC/NOX2 pathway as shown by positive correlation between the processes. The production of reactive oxygen species induced by stimulation of the FPR2 receptor is also increased, but it cannot be directly attributed to overexpression of the receptor mRNA or PKC/NOX2 activation, and requires further study. Understanding the mechanisms of regulation of the FPR1 and FPR2 signaling cascades may reveal new targets for anti-rheumatoid therapy.

Impact of Higher Calreticulin Gene Mutation Variant Allele Frequency in Patients with Myelofibrosis

Introduction. Calreticulin (CALR) mutations represent the second most frequent driver mut in myeloproliferative neoplasms (MPN), accounting for 20-30% of patients (pts) with Essential Thrombocythemia (ET) and 25-35% Primary Myelofibrosis (PMF). There are 2 types of CALR mutation, Type 1/like (T1), a del52 bp deletion and similar, and Type 2/like (T2), a 5bp insertion and similar, both in exon 9. Most patients have CALR variant allele frequency (VAF) below 50% (conventionally defined as heterozygous, He), while a few were reported with higher VAF (homozygous, Ho). Homozygosity, that may be associated to LOH at chr19, is phenotypically hallmarked by MPO deficiency in myeloid cells. While well-defined hematologic, clinical and prognostics correlates of the VAF of JAK2V617F mutation were reported in MPN, little information is available regarding CALR. Aim. The aim of the study was to characterize the hematological and clinical correlates of higher CALR VAF in a large population of CALR mut pts with primary (PMF) and PET-MF. Patients and Methods. All patients with a confirmed WHO2016 and IWG-MRT diagnosis of PMF and PET-MF, respectively, available in our database, who were CALR mutated, were included. CALR mut was assayed by PCR amplification and capillary gel electrophoresis starting from peripheral blood granulocytes. The VAF was determined by automated interpolation of the area under the curve, and expressed as the mutant peak area divided by (mutant peak area + wild-type peak area). Mutation analysis of a panel of 40 myeloid neoplasm-associated genes was performed by NGS (Oncomine, ThermoFisher). The nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimate of survival and log-rank test were used as appropriate. Results. A total of 121 pts (42 prePMF, 34.7%; 36 overt PMF, 29.8%; 43 PET-MF, 35.5%) were considered. Overall, T1 was found in 87 pts (71.9%), of which 8 (6.6%) were T1-like; T2 in 34 pts (28-1%), of which 3 (2.5%) T2-like. The median CALR VAF was 51.4+10.4%. For the purposes of the analysis, we considered at Higher VAF (CALR-h) pts having a VAF of >95% confidence interval of the distribution in the population, ie a VAF >55%, compared to Lower VAF (CARL-l; <55%). CALR-h was found in 28 pts, accounting for 23.1% of the study population. The frequency of T1 and T2 variants was similar in CALR-h and CALR-l pts, 71% and 28% and 71% and 29%, respectively. There was a significant trend of increased representation of CALR-h pts from prePMF (n=6, 14.3%) to overt PMF (n=8, 22.2%) and PET-MF (n=14, 32.6%); no difference in T1 and T2 relative representation in the different diagnostic category. ASXL1 mut were enriched among CALR-h pts (13/28, 46.4% vs CALR-l, 24/87, 27.6%; P=0.04) as it was the presence of any >1 mutation in an NGS-panel of myeloid genes (CALR-h 82.5% vs 57.5% in CALR-l, P=0.02). However, limited to HMR mutations, no difference was outlined. There was no significant difference between CALR-h and CALR-l pts as regards gender, age, IPSS score, karyotype abnormalities, BM fibrosis grade, constitutional symptoms, splenomegaly, thrombosis and bleeding events at diagnosis. On the other hand, CALR-h pts had lower Hb level (10.8 g/dl (8.3-14.6) vs 12.1 (7.4-16); P=0.02) and platelet count (466x109/L (102-1053) vs 661 (57-1800; P=0.02), and higher peripheral blood CD34+ cell counts (62.2x106/L (4-3450) vs 16.9 (0-2100); P=0.01). A greater proportion of CALR-h pts developed anemia during the FU (median 5.5y, range 0.3-33y). KM analysis revealed an anemia-free survival (Hb <10g/dL) of 7.9y (5.7-10y) in CALR-h vs 22.1y (13.9-30.3y) in CALR-l pts, P=0.004 (Figure). The HR (95%CI) was 3.1 (1.4-7.0). Also leukocytosis-free survival (leukocytes >25x109/L) was shorter in CALR-h pts (P=0.04), HR 2.55 (1.0-6.6). A CALR-h status did not impact on overall survival nor leukemia-free survival even after stratification based on disease subtype. Discussion. We found that about 20% of CALR mut MF pts harbor a higher VAF, here defined as VAF >55%, that is associated with a more anemic phenotype at diagnosis and greater risk of developing anemia in the FU; however, in spite of anemia development, overall survival was not significantly impacted in the different disease categories. Whether a CALR-h condition has different impact in ET remains to be assessed in a separate study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal