Granulocyte Fraction Research Articles

Tropoelastin modulates systemic and local tissue responses to enhance wound healing

Wound healing is facilitated by biomaterials-based grafts and substantially impacted by orchestrated inflammatory responses that are essential to the normal repair process. Tropoelastin (TE) based materials are known to shorten the period for wound repair but the mechanism of anti-inflammatory performance is not known. To explore this, we compared the performance of the gold standard Integra Dermal Regeneration Template (Integra), polyglycerol sebacate (PGS), and TE blended with PGS, in a murine full-thickness cutaneous wound healing study. Systemically, blending with TE favorably increased the F4/80+ macrophage population by day 7 in the spleen and contemporaneously induced elevated plasma levels of anti-inflammatory IL-10. In contrast, the PGS graft without TE prompted prolonged inflammation, as evidenced by splenomegaly and greater splenic granulocyte and monocyte fractions at day 14. Locally, the inclusion of TE in the graft led to increased anti-inflammatory M2 macrophages and CD4+T cells at the wound site, and a rise in Foxp3+ regulatory T cells in the wound bed by day 7. We conclude that the TE-incorporated skin graft delivers a pro-healing environment by modulating systemic and local tissue responses. Statement of significanceTropoelastin (TE) has shown significant benefits in promoting the repair and regeneration of damaged human tissues. In this study, we show that TE promotes an anti-inflammatory environment that facilitates cutaneous wound healing. In a mouse model, we find that inserting a TE-containing material into a full-thickness wound results in defined, pro-healing local and systemic tissue responses. These findings advance our understanding of TE's restorative value in tissue engineering and regenerative medicine, and pave the way for clinical applications.

Quantitative and Qualitative Composition of the Peripheral Blood of Fish in the Gradient of Long-Term Radiation Exposure

The paper deals with analysis of hematological parameters of the common rudd (<i>Scardinius erythrophthalmus </i>L.) and Prussian carp (<i>Carassius gibelio</i> Bloch) from the most radionuclide-contaminated water bodies of the Chernobyl Exclusion Zone. Data on absolute and relative leukocyte composition and morphological disorders of erythrocytes in peripheral blood were obtained. The leukocyte fraction of both fish species similarly responded to the absorbed dose rate. In the doses' range of 5.1-54.1 μGy/h, reactive changes of the compensatory nature were recorded with increase of the leukocytes content at the expense of lymphocyte and granulocyte fractions. Under the higher doses (up to 84.5 μGy/h), hematopoiesis was damaged with sharp decrease of the leukocytes' content and significant changes in leukograms, which was also confirmed by the hematological indices' values. The morphological disorders in erythrocytes increased along with absorbed dose rate.

Hematological parameters: is there a difference between those released by the hematological analyzer and to the customer?

This study aimed to compare the hematological parameters released by hematological analyzers with those released in customer reports. We conducted a descriptive study in the laboratories of a medium-sized municipality in the state of Minas Gerais registered in the National Register of Health Establishments. Interviews were conducted using a questionnaire to obtain information regarding the parameters released by the analyzers and those available in the customer's report. Sixteen laboratories were evaluated, and none of them released all the parameters obtained from the hematological analyzers to customers. The red blood cell distribution width was released in 88% of the laboratories, atypical lymphocytes in 70%, mean platelet volume in 50%, platelet distribution width and platelet count in 20%. No laboratory released information on reticulocytes, fraction of immature reticulocytes and immature granulocytes, nucleated erythrocyte count, immature platelet fraction and reticulocyte hemoglobin, and large platelet rate. All evaluated clinical analysis laboratories had at least one parameter that was not released in the customer's report despite being released by the hematological analyzers. The lack of knowledge on the part of professionals about the clinical importance of each parameter of the complete blood count results in a loss in patient assessment, and it is important to include these parameters in the complete blood count report.

Peculiarities of adaptational changes in leukocytes and platelets of yakutian ground squirrels during the pre-hibernation period in autumn

Earlier, it was shown that a series of physiological and biochemical changes in Yakutian long-tailed ground squirrels start to happen during autumn (the pre-hibernation period): in the brain, heart, skeletal muscles, etc. This study was conducted to analyze the composition of peripheral blood cells obtained from Yakutian ground squirrels in autumn, and for the first time an increase in the total number of leukocytes, particularly granulocytes, and a decrease in the number of lymphocytes were observed when compared with those obtained in summer; a shift of the leukogram from the lymphocytic fraction to the granulocytic fraction was revealed. Significant increase in a platelet count and thrombocrit with preservation of the percentage ratio of mature and young cell fractions was shown. Still, information on the cellular components of blood during the autumn season when hibernators prepare for winter months of hibernation remains scarce. Nonetheless, the results of our study indicate that there are very significant adaptive changes that occur in leukocytes and platelets of ground squirrels in this period. Possible causes of these changes are discussed.

Sonicate Fluid Cellularity Predicted by Transcriptomic Deconvolution Differentiates Infectious from Non-Infectious Arthroplasty Failure.

Although cellularity is traditionally assessed morphologically, deep sequencing approaches being used for microorganism detection may be able to provide information about cellularity. We hypothesized that cellularity predicted using CIBERSORTx (Stanford University), a transcriptomic-based cellular deconvolution tool, would differentiate between infectious and non-infectious arthroplasty failure. CIBERSORTx-derived cellularity profiles of 93 sonicate fluid samples, including 53 from subjects who underwent failed arthroplasties due to periprosthetic joint infection (PJI) (abbreviated for the purpose of this study as PJIF) and 40 from subjects who had undergone non-infectious arthroplasty failure (abbreviated NIAF) that had been subjected to bulk RNA sequencing were evaluated. Samples from PJIF and NIAF subjects were differentially clustered by principal component analysis based on the cellularity profile. Twelve of the 22 individual predicted cellular fractions were differentially expressed in the PJIF cases compared with the NIAF cases, including increased predicted neutrophils (mean and standard error, 9.73% ± 1.06% and 0.81% ± 0.60%), activated mast cells (17.12% ± 1.51% and 4.11% ± 0.44%), and eosinophils (1.96% ± 0.37% and 0.42% ± 0.21%), and decreased predicted M0 macrophages (21.33% ± 1.51% and 39.75% ± 2.45%), M2 macrophages (3.56% ± 0.52% and 8.70% ± 1.08%), and regulatory T cells (1.57% ± 0.23% and 3.20% ± 0.34%). The predicted total granulocyte fraction was elevated in the PJIF cases (32.97% ± 2.13% and 11.76% ± 1.61%), and the samples from the NIAF cases had elevated predicted total macrophage and monocyte (34.71% ± 1.71% and 55.34% ± 2.37%) and total B cell fractions (5.89% ± 0.30% and 8.62% ± 0.86%). Receiver operating characteristic curve analysis identified predicted total granulocytes, neutrophils, and activated mast cells as highly able to differentiate between the PJIF cases and the NIAF cases. Within the PJIF cases, the total granulocyte, total macrophage and monocyte, M0 macrophage, and M2 macrophage fractions were differentially expressed in Staphylococcus aureus compared with Staphylococcus epidermidis -associated samples. Within the NIAF cases, the predicted total B cell, naïve B cell, plasma cell, and M2 macrophage fractions were differentially expressed among different causes of failure. CIBERSORTx can predict the cellularity of sonicate fluid using transcriptomic data, allowing for the evaluation of the underlying immune response during the PJIF and NIAF cases, without a need to phenotypically assess cell composition.

Pediatric-Specific Patterns of Clonal Evolution Arising from Acquired Aplastic Anemia

Introduction Acquired aplastic anemia (aAA) occurs in both children and adults, though pathophysiology and clinical outcomes differ significantly by age. While clinical testing for paroxysmal nocturnal hemoglobinuria (PNH) clones is routine, targeted next generation sequencing (NGS) for myelodysplastic syndrome (MDS) associated variants has only recently been implemented. Furthermore, single nucleotide polymorphism array (SNP-A) assessing for copy neutral loss of heterozygosity in the human leukocyte antigen (HLA) region on chromosome 6p (6p CN-LOH) and HLA NGS assessing for allele specific loss of function (LoF) mutations, which together are seen in up to 25% of aAA cases, are only now being considered for clinical use. Recent studies have reported on the frequency and overlap of these 3 types of clonal evolution (PNH, MDS-associated, HLA-targeting) in adult aAA cohorts, but none focus specifically on pediatric patients, whose MDS-associated clonal evolution patterns are distinct. Delineating patterns of clonal hematopoiesis in pediatric aAA to determine whether evolution driven by immune escape (PNH and HLA loss) affects likelihood of MDS development is critical for personalizing therapeutic options. Thus, we aimed to describe the frequency, clinical significance, and overlap of clonal evolution including PNH, HLA loss, and MDS-associated gene mutations in a pediatric cohort of aAA patients. Methods This study identified 85 pediatric aAA patients treated at Children's Hospital of Philadelphia (CHOP) and enrolled on the IRB-approved CHOP/Penn BMF Registry and Repository in whom clinical testing for all patterns of clonal hematopoiesis was available or could be performed with biorepository samples. PNH flow cytometry was performed clinically in all cases; subjects with PNH > 0.05% in the granulocyte fraction were considered as having a PNH clone. Peripheral blood and bone marrow samples were used for SNP-A, HLA-NGS, and an institutional somatic mutation NGS panel for hematological malignancies encompassing 118 genes. Descriptive and contingency statistical analyses along with Fisher's Exact Test were performed using SAS (Cary, NC). Results Demographic composition of the 85 pediatric subjects with aAA included a median age of 10.7 years (1.7-20.8), male/female ratio of 59%/41%, and a representative race/ethnicity distribution (Table 1). In total, 66% displayed at least one clonal event. As expected, PNH clones were most common, present in 45% (38/85) of the cohort. An additional 16 (19%) exhibited acquired HLA loss by 6p CN-LOH (n=6), allele specific LoF mutations (n=6), or both (n=4). Fifteen (18%) had clonal hematopoiesis associated with MDS, of whom 3 had cytogenetic lesions that progressed to clinical MDS (3.5%), and 12 developed no morphologic features of MDS but demonstrated a total of 14 mutations in MDS-associated genes (BCOR/BCORL1 n=6, KMT2C/KMT2D n=3, RUNX1 n=1, ASXL1 n=1, SRSF2 n=1, PRPF8 n=1, CALR n=1). Critically, pediatric subjects with aAA who developed HLA loss did not develop MDS-associated mutations, with no patient demonstrating both subtypes of clonal events (Figure 1). In contrast, PNH and MDS-associated clonal events overlapped in 11% of the overall cohort. In total, 60% of patients with MDS-associated clonal hematopoiesis also possessed PNH clones, and 23% of patients with detectable PNH clones demonstrated at least one MDS-associated clonal event. Only 3.53% (n=3) of the cohort demonstrated both PNH and HLA loss, and PNH clone size was < 1% in all 3 patients. Furthermore, patients with HLA loss were significantly less likely to have PNH clones than those without HLA somatic alterations (19% vs 51%, p = 0.026). Correlations between patterns of clonal hematopoiesis and clinical outcomes will be presented. Concalusions Pediatric aAA patients treated with IST are at risk for developing MDS and hemolytic PNH. Presence and evolution of clonal hematopoiesis influenced decisions to pursue IST versus alternative donor transplant. Importantly, our study demonstrates that occurrence of clonal hematopoiesis by HLA loss does not increase and may even decrease the risk of developing MDS-associated or PNH clones. This finding strongly suggests that treatment decisions for aAA patients whose only clonal event is HLA loss need not be considered distinctly from patients with no clonal changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune cell composition of the bronchoalveolar lavage fluid in healthy and respiratory diseased dromedary camels

BackgroundRespiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry.ResultsCamel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals.ConclusionsCollectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

The effectiveness of percutaneous endoscopic lumbar discectomy combined with external lumbar drainage in the treatment of intervertebral infections.

ObjectiveTo analyze the effect of percutaneous endoscopic lumbar discectomy in treating lumbar intervertebral infections.MethodsA total of 13 patients with lumbar intervertebral infections who underwent percutaneous endoscopic lumbar discectomy combined with external drainage between November 2016 and December 2019 were enrolled in the present study. After the operation, sensitive antibiotics were used based on the results of the bacterial culture. If no pathogens were detected in the biopsy culture of the infected tissues, empirical antibiotics were administrated to these patients. The clinical efficacy was evaluated by using a visual analog scale (VAS), Japanese Orthopaedic Association (JOA), Oswestry Disability Index (ODI), and standard Macnab's evaluation. Postoperative computed tomography (CT) and MRI were also used to evaluate clinical efficacy.ResultsThe follow-up time was 10–18 months, and the average time was (13.69 ± 2.63) months. Causative bacteria were isolated in 7 of 13 infected tissue biopsy cultures. Systemic antibiotics and anti-tuberculous chemotherapy were administered according to sensitivity studies for identified. There were no pathogens isolated from the other six patients. Empiric antibiotics were administrated in these patients. One week after the operation, WBC, a fractional fraction of medium granulocytes, ESR and CRP were significantly lower compared to before the operation (all P < 0.05). At the last follow-up visit, the above-mentioned markers were all within normal range, which differed compared to the pre-operative data (P < 0.05). The VAS and ODI of the patients at 1 week and 3 months after operation were significantly lower compared to preoperative data (all P < 0.05). During the last follow-up visit, seven patients were excellent, five were good, and one was poor according to standard Macnab's evaluation. No serious complications were recorded.ConclusionsPercutaneous lumbar discectomy combined with external drainage resulted as an effective method for treating lumbar intervertebral infections and was associated with fewer injuries, less pain, low cost, and low recurrence rate.

The importance of immature granulocyte and immature reticulocyte fraction for the severity of acute bronchiolitis

The immature granulocyte (IG) and immature reticulocyte fraction (IRF) are new analytical parameters of the complete blood count, that have been studied as biomarkers of several inflammatory conditions. Here, our aim is to determine the effectiveness of IG and IRF percentages for the severity of acute bronchiolitis (AB). A single-center, prospective study was performed in patients who were hospitalized for acute bronchiolitis and healthy children were included as a control group. The demographic characteristics, white blood cell (WBC) count, platelet (PLT) count, eosinophil%, IG%, and IRF% values were analysed. Receiver operating characteristics (ROC) analysis was used to compare the diagnostic accuracies and predictive performances. We enrolled 168 infants in the acute bronchiolitis group and 80 in the control group. The Clinical Severity Score (CSS) showed that 48, 93, and 27 patients had mild, moderate, and severe bronchiolitis, respectively. The WBC, PLT, and IRF value were significantly higher in patients (p &lt; 0.001). There was no difference between the patients and control group in terms of IG and eosinophil percentage. Only a positive correlation was observed between the clinical severity of the AB and IRF (p=0.003).The ROC curve analysis indicated that the IRF% cut-off point for predicting severity AB was &gt;12.4, with a sensitivity of 53% and specificity of 88% (Areas under the curves (AUC):0.701,p&lt;0.001). The WBC count, PLT count, and IRF value increased in the AB group. The IRF percentage can be used to predict the clinical severity of AB in children.

Immature Granulocyte Percentage in Early Chronic Myeloid Leukemia

A 38-year-old man presented with mild fatigue of recent onset to the medical outpatient department (OPD). Clinical examination showed an absence of fever, pallor, and no organomegaly. Routine complete blood count (CBC) showed mild leukocytosis (total leukocyte count 22.93 × 109/L), absence of features of sepsis but with immature granulocyte (IG) fraction of 26.6% on peripheral blood film without basophilia or eosinophilia. A diagnosis of chronic myeloid leukemia (CML) was suspected which was confirmed by conventional karyotyping and quantitative reverse transcription − polymerase chain reaction (QPCR). Following administration of imatinib for 2 weeks, a follow-up CBC showed a reduction in IG fraction to 1.5%. The patient is under follow-up in OPD for 4 months and is responding well as substantiated by follow-up QPCR. To the best of our knowledge, this is the first ever report where CML was first suspected on the basis of IG fraction and the same parameter proved useful for follow-up.

Increased microchimerism in peripheral blood of women with systemic lupus erythematosus: relation with pregnancy.

We aimed to determine the presence, amount and origin of microchimerism in peripheral blood of pregnant and non-pregnant parous women with systemic lupus erythematosus (SLE) as compared to control subjects. We performed a comparative study in which peripheral blood was drawn from eleven female non-pregnant SLE-patients and 22 control subjects, and from six pregnant SLE-patients and eleven control subjects during gestation and up to six months postpartum. Quantitative PCR for insertion-deletion polymorphisms and null alleles was used to detect microchimerism in peripheral blood mononuclear cells and granulocytes. Microchimerism was detected more often in non-pregnant SLE-patients than control subjects (54.4% vs. 13.6%, respectively; p=0.03). When present, the median total number of foetal chimeric cells was 5 gEq/106 in patients and 2.5gEq/106 in control subjects (p=0.048). Microchimerism was mostly foetal in origin; maternal microchimerism was detected in one patient and one control subject. In control subjects, microchimerism was always derived from only one source whereas in 50% of patients it originated from multiple sources. The pregnant patients had a significantly higher median number of foetal chimeric cells in the granulocyte fraction just after delivery than control subjects (7.5 gEq/106 vs. 0 gEq/106, respectively; p=0.02). Just after delivery, SLE-patients had more microchimerism than control subjects. Three months post-partum, microchimerism was no longer detectable, only to reappear many years after the last pregnancy, more often and at higher levels in SLE-patients than in control subjects. This suggests that these chimeric cells may originate from non-circulating foetal chimeric stem cells.

ACE2 Is Expressed in Immune Cells That Infiltrate the Placenta in Infection-Associated Preterm Birth.

COVID-19 is associated with increased incidence of preterm birth (PTB). We assessed pathways by which SARS-CoV-2 could access the placenta. Placentae, from PTB with or without chorioamnionitis (ChA), or from term pregnancies (n = 12/13/group) were collected. Peripheral blood was collected from healthy pregnant women (n = 6). Second trimester placental explants (16–20 weeks, n = 5/group) were treated with lipopolysaccharide (LPS, to mimic bacterial infection) and ACE2, CCL2, IL-6/8 and TNFα mRNA was assessed. ChA-placentae exhibited increased ACE2 and CCL2 mRNA expression (p < 0.05). LPS increased cytokine and ACE2 mRNA in placental explants. Placental ACE2 protein localized to syncytiotrophoblast, fetal endothelium, extravillous trophoblast and in immune cells-subsets (M1/M2 macrophage and neutrophils) within the villous stroma. Significantly increased numbers of M1 macrophage and neutrophils were present in the ChA-placenta (p < 0.001). Subsets of peripheral immune cells from pregnant women express the ACE2 mRNA and protein. A greater fraction of granulocytes was positive for ACE2 protein expression compared to lymphocytes or monocytes. These data suggest that in pregnancies complicated by ChA, ACE2 positive immune cells in the maternal circulation have the potential to traffic SARS-CoV-2 virus to the placenta and increase the risk of vertical transmission to the placenta/fetus.

Telomere Length In Granulocyte, Mononuclear, CD34 and CD3 Cell Fractions In Primary Myelofibrosis and Age Matched Controls

BackgroundTelomeres are repetitive, noncoding DNA sequences that cap the ends of chromosomes to provide genome protection during cell division. Telomere shortening is a normal component of cellular aging and shortened telomeres are also associated with cancer. Leukocyte telomere lengths in patients with myeloproliferative neoplasms (MPN), especially in those with myelofibrosis (MF) and polycythemia vera (PV), have been shown to be shorter than normal controls and inversely correlate to JAK2V617F allele burden (Experimental hematology. 2013;41:627). There is inadequate information on the contribution of fractioned peripheral blood samples to the relative measurement of telomere length in MF, especially in the context of age-matched controls. MethodsPeripheral blood collected from each individual was subjected to density gradient centrifugation to enrich for granulocyte and mononuclear cell fractions. Antibody labeled magnetic bead separation was applied to a portion of the mononuclear cells to enrich for CD34+ and CD3+ fractions. DNA was isolated and telomere length was determined using quantitative PCR (qPCR) with primer sets specific to telomere repeat regions and a single copy gene. Relative telomere length of each sample was determined by calculating the Ct ratio of the telomere region and single copy gene. Quantitative PCR was used to measure JAK2V617F allele burden. ResultsThe study population included 20 patients with MF and 16 normal controls. Telomere length characterization in age-stratified normal controls indicated a clear effect of aging on telomere length, with significant differences for all cell fractions, especially when comparing age <50 years to age >65 years, and most pronounced for CD34-positive cells (Figure 1a; p=0.003). In MF, patients age 50-65 years displayed significantly shorter telomere lengths, in all cell fractions, compared to age-matched controls (Figure 1b; p=0.0001). The difference was most pronounced in CD34-positive cells (Figure 1b). However, older MF patients (age >65 years) did not differ in their telomere lengths of the aforementioned cell fractions, when compared to their age-matched controls; a trend for significance was seen for CD34-positive cells (Figure 1c; p=0.07). In MF, JAK2V617F allele burden was inversely correlated to telomere lengths of both granulocytes and mononuclear cells, in the younger (age 50-65 years; p=0.03) but not the older (age >65 years; p=0.76). [Display omitted] ConclusionsWe demonstrate the importance of age, age-matched controls, and specific peripheral blood cell fractions in the assessment of telomere length in MF. We identify CD34-positive cell fractions as the most reliable in assessing telomere length in MF but its value might be limited in patients older than age 65 years. It should be noted, however, that our results do not undermine the value of intra-patient comparison of telomere lengths, as might be necessary to monitor during treatment affecting telomerase activity. Disclosures:No relevant conflicts of interest to declare.

Modeling ASXL1 Mutation Revealed Myelodysplasia Caused By Derepression of p16Ink4a through Aberrant PRC1-Mediated Histone Modification

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant condition in patients with blood cells that possess somatic mutations of genes known to be recurrently mutated in hematologic malignancies. Among them is additional sex combs like 1 (ASXL1), whose mutation is frequent in various myeloid malignancies and associated with poor prognosis, and ASXL1 is known to act as an epigenetic modulator. Despite the clinical impact of ASXL1 mutations in myeloid malignancies, lack of animal models expressing mutated Asxl1 at endogenous levels mimicking human ASXL1-mutated diseases hinders from dissecting precise molecular mechanisms of mutated Asxl1. Here in this study, with newly generated knock-in mice carrying a frameshift mutation at the exon 13 of the Asxl1 gene (Asxl1G643WfsX12), which is identical to the most frequent human ASXL1 G646WfsX12 mutation, we extensively investigated the biological effects of the mutant.Asxl1G643fs/+ mice showed decreased body size and weight, resembling hypomorphic features in patients with Bohring-Opitz syndrome carrying de novo constitutive ASXL1 mutation.Asxl1G643fs/+ mice developed slowly progressive peripheral leukopenia, and Wright-Giemsa stain of bone marrow (BM) cells from Asxl1G643fs/+ mice showed dysplastic myeloid cells represented by hypersegmented neutrophils, apoptotic neutrophils and hyposegmented neutrophils with fine nuclear bridging consistent with pseudo-Pelger-Huët anomaly. Flow cytometric analyses of Asxl1G643fs/+ BM cells confirmed aberrant myeloid differentiation with significant increase of granulocytic and monocytic cell fraction, significant decrease of hematopoietic stem cell (HSC) pool (c-kit+ Sca-1+ Lin- (LSK) cells and CD34- CD135- LSK cells), increased apoptosis in hematopoietic stem/progenitor cells (HSPC) and significant accumulation of G1-phase HSC in the cell cycle analysis, all of which were reminiscent of human myelodysplastic syndromes (MDS). Interestingly, Asxl1G643fs/+ mice became moribund about 600 days after birth when compared to Asxl1+/+ littermates with anemia, leukocytosis, neutrophilia/monocytosis and hepatosplenomegaly, suggesting the development of MDS/myeloproliferative neoplasm (MPN)-like disease, most relevant to human cases with chronic myelomonocytic leukemia.RNA sequencing and gene set enrichment analysis in Asxl1G643fs/+ mice identified derepression of sets of genes that are normally repressed by BMI1, a critical regulator of histone modification in polycomb repressive comprex 1 (PRC1). Among these genes were p16Ink4a, p21, and p15Ink4b all of which were significantly upregulated in Asxl1G643fs/+ LSK cells. We performed chromatin immunoprecipitation (ChIP) assay with an anti-H2AK119ub1 antibody using LSK cells from Asxl1G643fs/+ mice and found that H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ LSK cells, suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. Co-immunoprecipitation assay and in vitro pull-down assay revealed that the mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild type ASXL1 protein. In addition, we performed ChIP assay with an anti-BMI1 and anti-FLAG antibody using a cell line stably expressed FLAG-tagged wild-type (WT) ASXL1 or mutant form (MT), and found that BMI1 level around the promoter region of p16Ink4a was significantly decreased in the ASXL1 MT-expressed cell line, whereas no significant change in the FLAG level between WT and MT. Furthermore, we intercrossed Asxl1G643fs/+ mice with p16Ink4a heterozygous knock out mice to elucidate the effect of p16Ink4a up-regulation in Asxl1G643fs/+ mice, and found restoration of HSC pool and amelioration of increased apoptosis in HSPC obtained from Asxl1G643fs/+p16Ink4a+/- mice. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in MDS-like phenotypes represented by decreased HSC pool and aberrant myeloid differentiation in Asxl1G643fs/+ mice.Taken together, our Asxl1 G643fs mutation knock-in mouse line is useful as a model of CHIP or early stage MDS, and our detailed analysis highlights the functional role of ASXL1 mutation on MDS and MDS /MPN development. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau.

A Non-Human Primate CRISPR/Cas9 Model of Clonal Hematopoiesis of Indeterminate Potential Demonstrates Expansion of TET2-Disrupted Clones

Recent large-scale genetic screening of human blood cells have identified that somatic mutations associated with clonal expansions commonly arise with aging, even in the absence of cytopenias, myelodysplasia, or leukemia. Loss of function or dominant negative mutations in genes encoding epigenetic modifier enzymes such as DNMT3, TET2, and ASXL1 are most common in this “clonal hematopoiesis of indeterminate potential (CHIP)”. However, a causal relationship between these mutations and clonal expansions or progression to myelodysplasia or leukemia is unclear, as it has been difficult to extrapolate from murine models due to the differences in lifespan, aging phenotypes and hematopoietic cell transformation susceptibility. We utilized CRISPR/Cas9 gene editing of hematopoietic stem and progenitor cells (HSPC) in the rhesus macaque model to study the relationship between mutations in these genes, and clonal expansion and progression to abnormal hematopoiesis.We optimized CRISPR/Cas9 gene-editing strategies to introduce loss-of-function mutations in rhesus macaque CD34+ HSPC and applied this methodology to macaque autologous HSPC transplantation. We initially focused on the DNMT3A gene, which has been reported as most frequently mutated in CHIP. Macaque CD34+ HSPC were split into three equal fractions, transduced with one of three lentiviral vectors carrying either a gRNA for the control AAVS1 locus, DNMT3A exon 3, or DNMT3 exon 19 (the location of the “hotspot” putative dominant negative mutation in CHIP), and then electroporated with Cas9 mRNA. These edited cells were then transplanted into the autologous macaque following ablative total body irradiation. A second macaque also received autologous CD34+ cells electroporated with either AAVS1- or DNMT3A-targeting Cas9 ribonucleoprotein (RNP) complexes. Both animals engrafted promptly, with detection of low level (0-4%) indels at the on-target sites for AAVS1 and DNMT3A for up to 12 months post transplantation, but so far, we have not detected clonal expansion of DNMT3A-mutated HSPC in these two animals.We next delivered Cas9 and a gRNA pool targeting DNMT3A, TET2, and ASXL1 (the top three mutated and expanded genes in CHIP) (80%) or AAVS1 (20%) into CD34+ HSPC, followed by autologous transplantation (Fig 1A). After engraftment, the level of indels at each target site in granulocytes was 2% or less. However, during the 12 months post-transplant, there was a gradual and marked expansion of the granulocyte fraction containing indels predicted to knock out TET2 function (Fig 1B). Of the 17 different indel signatures identified at 12 months, only two were over the limit of detection in the infusion product or at 1 month post-transplant (Fig 1C), suggesting that the expanding TET2 mutant clones originated from long-term HSPC rather than short-term progenitors or that rare, initially mutated clones were sufficient to initiate clonal expansions. Finally, TET2 mutation frequency in bone marrow CD34+ cells was more than 2-fold higher (~20%) than in mature granulocytes (8%) at 12 months post-transplant (Fig 1C), suggesting a possible block in differentiation or a defect in release from the marrow for TET2 mutated cells.In conclusion, we have successfully created a non-human primate model of CHIP utilizing autologous transplantation of CRISPR/Cas9-edited HSPC, demonstrating gradual expansion of TET2-mutated clones. In contrast, simple indel formation at the DNMT3A locus was not sufficient to result in clonal expansion in the three animals studied to date, and a strategy utilizing HDR to specifically knock-in the hotspot base pair recurrently mutated in CHIP will be required to further investigate the relationship of DNMT3A to CHIP. Taken together, these approaches should improve our understanding of the effects of TET2 loss of function on the development of CHIP and its link to hematologic and cardiovascular disease, as well as provide a model for the testing of preventative interventions for clonal expansion and progression to frank malignancy. [Display omitted] DisclosuresDunbar:Novartis/GSK to institute: Research Funding. Winkler:Novartis/GSK to institute: Research Funding.

Лейкоцитарный состав крови ужа обыкновенного Natrix natrix (Serpentes: Colubridae) Мордовского государственного заповедника (Россия)

The state of the grass snake (Natrix natrix) population in the Mordovian State Nature Reserve was assessed using the hematological approach. The leukocyte blood count was estimated together with calculation of the associated leukocyte indices (neutrophil:lymphocyte (N:L), eosinophil:lymphocyte (E:L), heterophil:eosinophil (H/E) and heterophil:lymphocyte (H:L) ratios). The surface area of leukocytes (heterophils, basophils, eosinophils, azurophils, monocytes, and lymphocytes) was measured. The leukocyte blood composition of Natrix natrix was characterized by the predominance of agranulocytes, and the granulocyte fraction was 31– 37% (u = 1.99, p = 0.04). The population pattern of the leukocyte blood composition of Natrix natrix was as follows: heterophils – 8.07±0.6%, basophils – 12.33±0.95%, eosinophils – 8.33±0.65%, azurophils – 5.25±0.53%, monocytes – 9.77±0.42%, and lymphocytes – 56.22±1.7%. According to the averaged area, the leukocytes were arranged in the following order: eosinophils – monocytes – azurophils, basophils – heterophils – lymphocytes. The diameter of the largest cells (eosinophils) was (17.5±2.42) μm. The smallest lymphocytes had diameters within (7.22±1.21) μm. The absence of any differences in the quantitative content of all types of granulocytes and agranulocytes in the blood of the grass snake from different parts of the reserve indicated a comparable level of impact and the identity of the physiological mechanisms of adaptation that occur in the body of animals in protected habitat conditions. Most of the leukocyte integral indices revealed the same type of variability in the parameters of the white blood cell system of the Natrix natrix in three areas of the reserve, with the exception of an increased value of the heterophiles/lymphocytes ratio in the sample from the village Pushta. The increase in this indicator made it possible to draw a conclusion about the stress effect on the individuals of this sample. The principal component method was used to differentiate the samples of the grass snake with a more complete quantitative description of the leukocyte composition of the objects of study; the results obtained are presented in a visual, integrated and generalized form. The use of the principal component method made it possible to combine samples of Natrix natrix with similar indicators of leukocyte composition, as well as to identify a population of Natrix natrix that differs in its indicators in the urbanized territory of the Samara region. The blood pattern and the dynamics of blood leukocyte indices reflected the active response of the Natrix natrix body to the complex of environmental factors of the habitat on the territory of the Mordovian State Nature Reserve.

Dynamic Leukocyte Populations Are Associated With Early- and Late-stage Lesions in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition typically targeting the axillary and anogenital regions of the body. The massive inflammatory cell infiltrate produced in this cryptogenic condition has led investigators in the attempt to link particular inflammatory cell fractions and cytokines to disease development, and ultimately to disease treatment. This study qualitatively and quantitatively analyzes the white blood cell fractions of macrophages, B-lymphocytes, T-lymphocytes, plasma cells, and granulocytes in 104 HS lesions on formalin-fixed paraffin-embedded tissues using immunohistochemistry (IHC). Four dermis-associated epithelial categories were investigated from persons with HS: 15 unaffected HS skin (US), 19 distended but unruptured follicle epithelium (UF), 62 migrating stratified squamous epithelium (MSSE) from ruptured follicles, and 35 degraded migrating epithelial sheets (DMES). In addition, 27 control skin (CS) from persons without HS were evaluated. Analysis of cell counts indicated that non-migratory dermal epithelium (CS, US, and UF) stimulated very little inflammatory response. However, contrary to previous studies which indicated macrophages to be the chief inflammatory cell in HS, this study showed that plasma cells were the primary cell type present in early-stage HS lesions (MSSE), whereas granulocytes were the major cell population seen in late-stage HS lesions (DMES).

Immature cell fractions after cessation of chronic P2Y12-inhibition in patients with coronary artery diseases

Changes in circulating cell populations may promote ischemic events that occur soon after discontinuation of P2Y12-inhibition. The aim of the study was to track the course of thrombopoietic and erythropoietic cells in patients with coronary artery diseases (CAD) after planned and physician-driven cessation of chronic P2Y12-inhibition (clopidogrel 75 mg OD, or prasugrel 10 mg OD, or ticagrelor 90 mg BID). Cell fractions were determined in 62 patients at baseline (the last day of P2Y12-inhibitor intake), on day-10, day-30, and day-180 thereafter. Immature platelet fraction (IPF), immature reticulocyte fraction (IRF), reticulocyte hemoglobin content (Ret-Hb) and red blood cell count (RBC) significantly increased from baseline to day-180 (IPF: p = .003; IRF: p = .013; Ret-Hb: p < .001; RBC: p = .044). Platelet count, leucocyte count and immature granulocyte fraction did not change over time (p = .561, p = .869, and p = .161, respectively). Fibrinogen levels significantly declined over time (p = .011), thrombopoietin levels increased in a non-significant manner (p = .379). We did not observe any significant interaction with choice of P2Y12-inhibitor, therefore suggesting a drug class-effect. Our data shows, that discontinuation of dual antiplatelet therapy is associated with raised thrombopoietic and erythropoietic activity in the bone marrow, without significant upregulation of thrombopoietin. This provides further evidence for a direct stimulation of precursor cells by P2Y12-inhibitors.

Silibinin Upregulates CXCR4 Expression in Cultured Bone Marrow Cells (BMCs) Especially in Pulmonary Arterial Hypertension Rat Model.

Previously we reported that silibinin ameliorated pulmonary arterial hypertension (PAH) in rat PAH models, possibly through the suppression of the CXCR4/SDF-1, until the point where PAH became a severe and irreversible condition. To further investigate how silibinin ameliorates PAH, we first attempted to clarify its effect on bone marrow cells (BMCs), since the CXCR4/SDF-1 axis is known to regulate stem cell migration and attachment in BM niches. Rat PAH models were established through a combination of a single subcutaneous injection of monocrotaline (MCT) and chronic hypoxic conditions (10% O2). BMCs were harvested and cultured, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were performed to investigate whether silibinin affected CXCR4 expression. Silibinin upregulated the gene expression of stem cell related markers CXCR4, SDF-1, SCF, and c-Kit, inflammatory markers IL-6 and TNFα, mesenchymal stem cell (MSC)-related markers CD44 and CD29, and the granulocyte/monocyte-macrophage marker CD14 in cultured BM in PAH rats, but not in normal rats, except CXCR4. FCM showed that silibinin increased the CXCR4-positive cell population in a granulocyte fraction of cultured BMCs. However, immunohistochemical (IHC) staining showed no significant change in CXCR4 expression in the BM of the tibias. These results suggest that silibinin increases the expression of CXCR4 in BM, and the increased CXCR4-positive cells could be granulocytes/monocyte-macrophages.