Immature Neutrophils Research Articles

Characterization of Zymosan-Modulated Neutrophils With Neuroregenerative Properties.

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines—THP-1 and Mono Mac 6—Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

Alveolar rhabdomyosarcoma with massive bone marrow infiltration and disseminated intravascular coagulation mimicking acute leukemia.

A 27-year-old man was admitted with a 2-month history of general fatigue, appetite loss, and back pain, leading to a worsening of his general condition. On admission, laboratory tests showed an increased white blood cell count (15.9 × 109/L), with leucoerythroblastosis (myeloblasts, 1.0%; promyelocytes, 0%; myelocytes, 2.5%; metamyelocytes, 1.5%; band neutrophils, 3.5%; segmented neutrophils, 67.0%; lymphocytes, 18%; erythroblasts, 48%), severe anemia (hemoglobin, 45.0 g/L), and thrombocytopenia (platelet count, 21.0 × 109/L). Biochemical tests revealed elevated lactate dehydrogenase (2582 U/L) level, and coagulation tests showed a prolonged prothrombin time (20.3 s; reference interval: 10.0–13.0), normal fibrinogen (2.24 g/L), elevated fibrinogen-fibrin degradation product (331.9 mg/L), plasmin inhibitor-plasmin complex (9.05 mg/L), thrombin-antithrombin complex (41.2 μg/L), and decreased antithrombin (75.3%) levels. A computed tomography scan revealed systemic lymphadenopathy, splenomegaly, bilateral pleural effusions, and massive ascites. A bone marrow aspirate revealed massive infiltration by large abnormal cells with a basophilic cytoplasm and cytoplasmic vacuoles (Figure 1A). Flow cytometry analysis showed that the abnormal cells expressed only CD56, but no myeloid or lymphoid cell markers. Because he suffered severe disseminated intravascular coagulation (DIC) and worsening hypoxemia due to increased pleural effusions, initial treatment for acute leukemia comprised prednisolone and cytarabine. On Day 2 postadmission, biopsy of the right axillary lymph node was performed. Immunohistological findings from a bone marrow clot and biopsy tissue from the right axillary lymph node revealed diffuse proliferation of abnormal round cells, which expressed desmin (Figure 1B), myogenin, vimentin, and CD56 (Figure 1C), but not cytokeratin and leukocyte antigen. Therefore, a diagnosis of alveolar rhabdomyosarcoma was made. Subsequently, treatment with vincristine, doxorubicin, and cyclophosphamide was initiated, which ameliorated the lymphadenopathy, ascites, and DIC. Although a leukemic phase or bone marrow infiltration is an uncommon manifestation of alveolar rhabdomyosarcoma, flow cytometry and immunohistological analyses of bone marrow specimens can facilitate correct diagnosis. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors received no specific funding for this work. AS, HK, HU, and SK contributed to patient's care. SK performed pathological diagnosis. All authors wrote and reviewed the manuscript. Written informed consent for publication was obtained from the patient. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent for publication was obtained from the patient. All data can be assessed by contacting the corresponding author.

Gastric Ulcers in Alpacas- Clinical, Laboratory, and Pathological Findings.

Gastric ulcers are a common finding in post-mortem examinations of South American camelids (SAC), but diagnosis in living animals is often difficult. The aim of this study was to provide an overview of the incidence of gastric ulcers in alpacas, common concomitant diseases, and clinical as well as laboratory findings to facilitate diagnosis for veterinarians. For this purpose, a total of 187 necropsy reports of alpacas were evaluated, including clinical and laboratory findings on the living animal. A total of 23.5% of the animals (n = 44) were found to have gastric ulcers, nine were perforated. Compartment 3 was most frequently affected by gastric ulcers. No sex predilection could be detected, but animals 1 year of age and older were more frequently affected by gastric ulcers than animals under 1 year of age. Alpacas with gastric ulcers were presented to the clinic due to different non-specific symptoms. In alpacas with gastric ulcers, significantly more organs or organ systems besides the stomach revealed clinical findings than in animals without gastric ulcers. Of the 44 animals with gastric ulcers, a total of 21 alpacas (47.7%) had a poor nutritional status, but cachexia was not significantly more frequent in animals with gastric ulcers than in other dissected animals without ulcers. Hematologic investigations revealed a significantly lower white blood count and significantly lower segmented neutrophils than in deceased animals without ulcers. Compared to animals discharged after treatment, alpacas that died with gastric ulcers had significantly higher levels of band neutrophils and fewer eosinophils and basophils. Occult blood in feces was found in three of 12 animals with gastric ulcers examined for occult blood. In summary, gastric ulcers are a common problem in SAC, which is difficult to diagnose clinically or by laboratory investigations. As these are often chronic processes involving other organ systems, regular monitoring of the animals' nutritional status and early detection of disease symptoms may help to prevent gastric ulcers.

First Estimates of Age and Growth of the Lusitanian Cownose Ray (Rhinoptera marginata) from the Mediterranean Sea

Within the eastern Mediterranean, increased fishing pressure coupled with a lack of basic biological information is threatening the sustainability of the rare Lusitanian cownose ray (Rhinoptera marginata). To provide baseline life-history data for this species, age estimates were obtained from vertebral band counts of 224 rays (size range: 210–998 mm disc width and 140 to 13,600 g weight) collected from Iskenderun Bay. Females ranged in size from 210 to 998 mm disc width and 238 to 13,600 g weight, while males ranged from 130 to 866 mm disc width and 140 to 8250 g weight. The index of average percent error (2.8%) and age-bias plot suggest that the aging method used represents a precise and non-biased approach. Marginal increment analysis indicated that a single opaque band is deposited annually between August and September. The oldest ages obtained for R. marginata were 9 years for males and 19 years for females, which corresponded to total lengths of 866 and 998 mm disc width, respectively. For males, limited samples prevented the accurate calculation of growth rates; however, for combined sexes, observed and disc width-at-age data resulted in the following von Bertalanffy growth parameters: DW∞ = 1102.16 mm, k = 0.148 and t0 = −0.2167. Although additional samples are necessary to determine growth rates in males, the results of the present study indicate that R. marginata females exhibit life history characteristics similar to other Rhinoptera species.

Utility and prognostic significance of leukocyte ratios in dogs withPrimary Immune-Mediated Hemolytic Anemia.

Canine immune-mediated hemolytic anemia (IMHA) is a life-threatening condition that is commonly associated with neutrophilia and monocytosis. Leukocyte ratios have been found to have prognostic value in humans and animals affected by a range of inflammatory, infectious, and neoplastic disorders. We hypothesized that in primary IMHA, neutrophil to lymphocyte (NLR), neutrophil to monocyte (NMR), band neutrophil to segmented neutrophil (BNR) and monocyte to lymphocyte (MLR) ratios would be higher in dogs that did not survive to discharge. Medical records of dogs diagnosed with IMHA at two veterinary teaching hospitals were retrospectively reviewed. Twenty-three of the 72 included dogs did not survive to discharge. NLR, NMR, BNR and MLR ratios were compared between dogs that survived to discharge and dogs that died or were euthanized. None of the ratios were significantly different between survivors and non-survivors (P = 0.14-0.99). Area under the Receiver Operating Characteristic (ROC) curve for prediction of non-survival ranged from 0.5 (95% confidence interval 0.38-0.62) for MLR to 0.61 (0.49-0.72) for NMR and was not significantly different from 0.5 for any ratio (P = 0.29-0.99). After exclusion of 31 dogs that received one or both immunosuppressive medications and blood transfusion before presentation, the area under the ROC curve for prediction of survival was significantly different from 0.5 for MLR (0.78, P = 0.01) and NMR (0.78, P = 0.0002). This study suggests that lower MLR and higher NMR may predict poorer prognosis in untreated dogs with IMHA.

Androgen exposure alters the neutrophil response to pyelonephritis

Abstract Urinary tract infections (UTIs) in men are uncommon but carry increased risk for severe upper-tract UTI (pyelonephritis) and complications including renal scarring. In preclinical models of Escherichia coli UTI, male C3H/HeN mice uniformly develop high-titer pyelonephritis (most with renal abscesses), while the majority of female C3H/HeN mice resolve renal infection within 7–14 days. We previously showed that this difference in outcomes is testosterone dependent. Here, we find that androgen exposure in female C3H/HeN mice alters the neutrophil response to UTI, increasing the propensity for severe renal inflammation and abscess formation as opposed to bacterial clearance and resolution of infection. Concordant with our prior published results, androgen exposure in C3H female mice given Escherichia coli UTI led to 3-log higher bacterial loads in the kidneys by 14 days post infection. We found that the kidneys of androgen-exposed mice harbored more neutrophils than standard females at baseline and throughout infection, correlated with higher levels of the neutrophil-recruiting chemokine CXCL1 (murine KC) in kidney tissue. In addition, the kidneys of androgen-exposed females were enriched for aged, immature neutrophils (Ly6G+, CD49d+, CD101−). Compared to their mature (CD101+) counterparts, these cells exhibited markers of increased degranulation, altered phagocytic activity, and diminished capacity for efflux from kidney tissue. These data support a model in which androgen-exposed mice, despite an apparently more robust neutrophil presence, fail to control renal bacterial infection due to altered neutrophil functions. Supported by grants from NIH (R01 DK111541, R01 DK126697, R01 AI158418)

A Distinct Immature Low-Density Neutrophil Population Characterizes Acute Generalized Pustular Psoriasis

Generalized pustular psoriasis (GPP) is a severe disease featured by neutrophilic pustules and enhanced IL-36 inflammatory pathway in the skin (Hoegler et al., 2018). PBMCs reflecting the systemic inflammatory activation can provide pathogenetically relevant information (Swindell et al., 2016). Recently, a transcriptomic analysis of PBMCs and neutrophils in patients with GPP has been performed in a stable disease state (Haskamp et al., 2021). However, transcriptomic profiling of PBMCs during an acute flare of GPP is unclear.

Disseminated Talaromyces marneffei in newly diagnosed HIV patient.

A 31-year-old Chinese man referred to the haematology clinic, was presented with recurrent fever for 19 days and two enlarged lymph nodes on the left neck. Past medical history was unremarkable. Laboratory data revealed white blood cell (WBC) count 9.81 × 109/L (neutrophilic band granulocyte 67%, neutrophilic myelocyte 11%, neutrophilic metamyelocyte 7%, polymorphonuclear neutrophil 6%, lymphocyte 5%, eosinophil 1%, monocyte 2%, basophil 1% and nucleated red blood cell 18/100 WBC), haemoglobin 77 g/L and platelet count 41 × 109/L. The peripheral blood and bone marrow aspirate smear revealed small round-to-ovoid yeast cells located predominantly within or surrounding the neutrophil (top left, middle and right, 1000×, Wright-Giemsa staining). The blood culture showed the fungus grown in a mould form arranged in a mulberry-like pattern and produced a soluble red pigment that diffuses into the sabouraud medium (bottom left). Lactophenol cotton blue staining confirmed numerous yeast-like organisms by the microscopic morphology (bottom middle, 400×). Fungus staining also showed numerous blue yeast-like organisms by fluorescence microscope (bottom right, 400×). The patient was subsequently diagnosed with AIDS having HIV antibody, and the blood cultures showed the growth of Talaromyces marneffei, a dimorphic fungus that is an important opportunistic pathogen. After treatment with anti-fungus and anti-HIV drug, this patient had significantly improved and discharged. The authors would like to thank the encouragement and support of the members who take advanced study in the unit of cytology of the Department of Clinical Laboratory, Zhejiang Provincial People's Hospital. Yanxia Chen was supported by the Zhejiang Provincial Natural Science Fund (No. LY20H100008). Financing entity had no role in the study design, data collection or analysis, decision to publish or preparation of the manuscript. The authors declared that they have no conflict of interest. Zhejiang Provincial Natural Science Fund, Grant Number: LY20H100008. Yingcai Huang provided the haematology picture and wrote the manuscript. Haiyan Cui provided the microbiology picture. Yanxia Chen analysed the data. Jinlin Liu wrote the manuscript. All authors reviewed this manuscript and agreed to submit it. This study was approved by the ethical committee of Longgang District People's Hospital of Shenzhen & The Second Affiliated Hospital of the Chinese University of Hong Kong (No. 2022007). This study did not involve any personal information, but only reports the laboratory data. Therefore, the patient consent was waived, and this waive was approved by the ethical committee of Longgang District People's Hospital of Shenzhen & The Second Affiliated Hospital of the Chinese University of Hong Kong.

Hyperspectral-Based Classification of Managed Permanent Grassland with Multilayer Perceptrons: Influence of Spectral Band Count and Spectral Regions on Model Performance

Detailed knowledge of botanical composition is a key factor for the sustainable and site-specific management of permanent grassland and facilitates an improvement in the performance and efficiency of livestock feeding. Spectral-based data acquisition combined with machine learning has the potential to classify species groups and plant parts in permanent grassland with high accuracy. However, a disadvantage of this method is the fact that hyperspectral sensors with a wide spectral range and fine spectral and high spatial resolution are costly and create large amounts of data. Therefore, the question arises as to whether these parameters are necessary for accurate grassland classification. Thus, the use of sensors with lower spectral and spatial resolution and correspondingly lower data processing requirements could be a conceivable approach. Therefore, we investigated the classification performance with reduced predictor sets formed by different approaches in permanent grassland. For pixel-based classification, a cross-validated mean accuracy of 86.1% was reached using a multilayer perceptron (MLP) including all 191 available predictors, i.e., spectral bands. Using only 48 high-performing predictors, an accuracy of 80% could still be achieved. In particular, the spectral regions of 954 nm to 956 nm, 684 nm to 744 nm and 442 nm to 444 nm contributed most to the classification performance. These results provide a promising basis for future data acquisition and the analysis of grassland vegetation.

Data-Driven EEG Band Discovery with Decision Trees.

Electroencephalography (EEG) is a brain imaging technique in which electrodes are placed on the scalp. EEG signals are commonly decomposed into frequency bands called delta, theta, alpha, and beta. While these bands have been shown to be useful for characterizing various brain states, their utility as a one-size-fits-all analysis tool remains unclear. The goal of this work is to outline an objective strategy for discovering optimal EEG bands based on signal power spectra. A two-step data-driven methodology is presented for objectively determining the best EEG bands for a given dataset. First, a decision tree is used to estimate the optimal frequency band boundaries for reproducing the signal’s power spectrum for a predetermined number of bands. The optimal number of bands is then determined using an Akaike Information Criterion (AIC)-inspired quality score that balances goodness-of-fit with a small band count. This data-driven approach led to better characterization of the underlying power spectrum by identifying bands that outperformed the more commonly used band boundaries by a factor of two. Additionally, key spectral components were isolated in dedicated frequency bands. The proposed method provides a fully automated and flexible approach to capturing key signal components and possibly discovering new indices of brain activity.

Extracellular Traps Increase Burden of Bleeding by Damaging Endothelial Cell in Acute Promyelocytic Leukaemia.

The rate of complete remission of acute promyelocytic leukemia (APL) is currently over 90% because of the use of all-trans retinoic acid (ATRA) with arsenic trioxide (ATO). However, hemorrhagic mortality has emerged as the most significant barrier to APL-induced remission. Neutrophils extracellular traps (NETs/ETs) cause vascular leakage by damaging the integrity of endothelial cells. We have previously demonstrated that APL cells treated with ATRA/ATO undergo a cell death process, releasing extracellular chromatin, termed ETosis/NETosis. However, the mechanism underlying the involvement of ETs in endothelial injury in APL remain largely unknown. Here, we analysed the ability of mature and immature neutrophils to release ETs, and their interaction with platelets (PLTs) in APL. Importantly, the effect of ETs on vascular endothelium in APL was discussed. Our results showed that the ability of immature neutrophils to release ETs was impaired in APL, whereas mature neutrophils produced ETs, which were associated with activated PLTs. Moreover, ATRA+ATO induced immature neutrophil differentiation, as well as increased the release of ETs from mature neutrophils. The excessive ETs damaged endothelial cells, causing blood cell leakage. Removing ETs using DNase 1 alleviated endothelial damage and improved blood cells leakage. Our results indicate that vascular endothelial injury is at least partially associated with ETs in APL, and that targeting ETs production may be an effective approach for relieving vascular leakage and reducing the burden of bleeding in APL.

Diagnostic utility of combined immature and total neutrophil counts along with C-reactive protein in early detection of neonatal sepsis: A cross-sectional study

IntroductionA timely diagnosis is critical for management of Neonatal sepsis. Blood Culture is considered to be the “Gold Standard” for its diagnosis, but it has some limitations. In recent times, highly sensitive and specific inflammatory markers like interleukins, ELISA, counter immune-electrophoresis etc. have been in use for its diagnosis. But these are impractical for developing countries, due to their high cost and requirement of sophisticated equipments. A combination of haematological parameters like total leucocyte count (TLC), immature to total neutrophil ratio (I/T ratio), absolute neutrophil count (ANC), platelet count and C-reactive protein (CRP) estimation provide an early diagnosis of bacteremia. This study was undertaken to evaluate the usefulness of the above mentioned parameters as indicators for early diagnosis of neonatal sepsis. Material and methodsIn the present cross-sectional study, we intent to analyse various hematologic parameters in 160 neonates admitted in the neonatal care unit of a tertiary care hospital in Delhi. We obtained data from the records of blood culture and complete blood counts of neonates from pathology and microbiology departments of the hospital. Out of 160 admitted neonates, 80 were taken as cases and remaining 80 were taken as controls. Medical records were studied to identify infants born at ≥ 34 weeks gestation. CBCs was analysed, blood cultures and CRP were done in department of Microbiology. CBC, CRP and Blood culture was done as per standard protocols and clinical assesment by paediatrician. The statistical analyses were performed using SPSS version 22 for windows. ResultsAmong 80 neonates, who were in early neonatal sepsis, 44 cases (55%) were females, and 36 (45%) were males. The Microbiological profile of 80 septic neonates was analysed. The I/T value, ANC and CRP values were significantly higher in the neonates suffering from sepsis as compared to the control group. Among 80 septic neonates (cases), 30 (37.5%) were having normal ANC while 50 (62.5%) were having increased ANC and 34 (42.5%) were having normal I:T ratio while 46 (57.5%) were having increased I:T ratio. Out of 80 septic neonates (cases), 18 (22.5%) were having normal CRP while 62 (77.5%) were having increased CRP. ConclusionANC, I/T Ratio and CRP are quick, simple and cost-effective routine laboratory tests which help in early diagnosis of neonatal sepsis. Although there are many serological markers available, ANC and I/T Ratio serves as a reliable predictor of neonatal sepsis. With a good sensitivity, high specificity and a good negative predictive value these parameters can therefore help in timely and early identification of neonatal sepsis.

Absolute band neutrophils count is a predictor of overall survival in advanced uterine cervical cancer.

Neutrophils play a role during the oncogenic process, and their count can be a prognostic marker. The objective was to evaluate the association of band and segmented neutrophils with prognostic factors, overall survival, and disease-free survival in advanced uterine cervical cancer (IIB to IVB staging). This study evaluated 88 patients diagnosed with uterine cervical cancer staging IIB to IVB. The recorded data from medical records were age, parity, staging, histological type, absolute count of total neutrophils, band neutrophils and segmented neutrophils, disease-free survival, and overall survival. Receiver-operating characteristic (ROC) curve was used to obtain the area under the curve (AUC) and determine the best cut-off values for each parameter. Survival was verified by the Kaplan-Meier method, and multivariate analysis was performed by Cox regression. The level of significance was ≤ 0.05. Regarding the total neutrophils, band, and segmented neutrophils count, a cut-off value of 6187/mm3, 273 mm3 and 6062/mm3 were found, respectively. Overall survival was shorter in patients with total neutrophils greater than 6187/mm3 (p = 0.012), band neutrophils greater than 273/mm3 (p = 0.001), and segmented neutrophils greater than 6062/mm3 (p = 0.013). After multivariate analysis considering the two types of neutrophils, only band neutrophils greater than 273/mm3 remained as an independent factor for shorter overall survival. The absolute count of band neutrophils greater than 273/mm3 was a potential predictor of shorter overall survival in women with invasive cervical cancer. This count can be of great clinical use, in addition to being inexpensive, less invasive, and easy to perform.

Interleukin-17E, inducible nitric oxide synthase and arginase1 as new biomarkers in the identification of neutrophilic dermatoses.

SummaryBackgroundNeutrophilic dermatoses (ND) are a heterogeneous group of diseases, but can often have a relatively similar histological appearance.AimTo identify a combination of biomarkers allowing a better differentiation of ND types.MethodsBiopsies were obtained from normal human skin (NS; n = 4), chronic plaque‐type psoriasis (PsO; n = 7), paradoxical psoriasis (PP; n = 8), generalized pustular psoriasis (GPP; n = 9), subcorneal pustular dermatosis of Sneddon–Wilkinson (SPD; n = 3), acute generalized exanthematous pustulosis (AGEP; n = 3), hidradenitis suppurativa (HS; n = 7), Sweet syndrome (SS; n = 8) and pyoderma gangrenosum (PG; n = 8). Samples were analysed by immunofluorescence using three biomarkers, interleukin (IL)‐17E, inducible nitric oxide synthase (iNOS) and arginase1, each one in combination with two cell markers, myeloperoxidase (MPO) and CD68, which allow the identification of neutrophils and macrophages, respectively.ResultsWe found that SS is characterized by high expression of IL‐17E and iNOS in the epidermis, while PG exhibits low expression. The density of the neutrophil infiltrate helps to differentiate PP (high‐density infiltrate) from PsO (low‐density infiltrate). High expression of arginase1 in the granular layer of the epidermis is a hallmark of SPD. Finally, mature neutrophils and proinflammatory macrophages are readily detectable in PP, SPD and PG, whereas immature neutrophils and anti‐inflammatory macrophages are more frequent in GPP, AGEP, HS and SS.ConclusionsThe analysis of ND by immunofluorescence using IL‐17E, iNOS and arginase1 in combination with MPO and CD68 allows for characterization of differential expression patterns in the epidermis as well as the determination of the polarization status of the dermal neutrophils and macrophages. The appropriate markers may help in the differentiation of ND in clinical practice.

B01: STROMAL CELL-NEUTROPHIL INTERACTIONS PROMOTE A PRO-TUMOR ENVIRONMENT IN MULTIPLE MYELOMA

Cancer development and progression are accompanied by alterations in the local microenvironment. In multiple myeloma (MM), interactions between myeloma cells and their niche are considered critical for disease pathobiology. Recently, we showed that the MM bone marrow (BM) is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors such as IL6, as well as myeloid cell modulation factors such as CCL2, ANXA1, C3 and chemokines that bind CXCR1 and 2. As myeloid cells have been implicated in the pathophysiology of various malignancies, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. Using flow cytometry, we observed increased expression of CXCR1/2 on CD15+ neutrophils in MM compared to those of controls. In addition, BM neutrophils in MM were activated, as evidenced by a switch to the active form of CD11b and the shedding of CD62L. As these findings suggested possible neutrophil – iMSC interactions, we set out to identify MM-associated alterations in neutrophils by scRNA sequencing of the full BM granulocytic lineage (n = 6 MM, and 4 controls). Immature and mature neutrophils in MM had increased transcription of genes encoding receptors for iMSC-derived signals, including IL6R, FPR and C3AR1. Moreover, we confirmed the activated state of neutrophils through elevated transcription of OSM, SLPI, and IL1B. These data suggest a contribution of iMSCs to neutrophil activation. This was confirmed by coculture of iMSCs and naïve neutrophils, which lead to shedding of neutrophilic CD62L. Further analysis of our transcriptional data revealed that a subpopulation of activated MM neutrophils expressed interferon (IFN)-response genes, including IFIT1, IFIT2 and ISG15. Importantly, by analyzing single cell immune datasets of MM bone marrow, we observed IFN-responsive neutrophils to be the only hematopoietic population transcribing TNFSF13B, encoding the MM-survival factor BAFF. Stimulation of naïve neutrophils with IFNγ or IFNβ induced TNFSF13B transcription, but did not lead to BAFF secretion. This led us to hypothesize that BAFF release by activated neutrophils might be regulated by the inflammatory stromal environment. To test this hypothesis we cultured IFNγ-stimulated neutrophils in the presence of either non inflammatory MSCs or IL1β-activated iMSCs. Whilst the presence of iMSCs could induce BAFF release by neutrophils, non-inflammatory MSCs did not have this effect. Together these data suggest that stromal – immune interactions in MM are driving a tumor-supportive environment by inducing local release of plasma cell survival factors.

ПРИНЦИПЫ НАЗНАЧЕНИЯ ГЛЮКОКОРТИКОСТЕРОИДОВ У ПАЦИЕНТОВ С COVID-19

Introduction. This article discusses the treatment of coronavirus infection (COVID-19) with glucocorticoste- roid drugs (GCS), side effects of drugs and their prevention, transfer from intravenous to intramuscular and then to oral administration, and the development of withdrawal syndrome. The article describes the conditions under which antibiotic therapy is prescribed, as well as the conditions under which the combined use of corticosteroids and antibiotics is neces- sary. Aim. The aim to analyze the basic principles of corticosteroids prescribing in the treatment of COVID-19: indications for corticosteroids administration, administration regimens and required dosages, side effects of corticosteroids admin- istration. Material and methods. The article uses data from the Interim Guidelines for the Prevention, Diagnosis and Treatment of New Coronavirus Infection (COVID-19), the Federal Clinical Guidelines for the Specialty “Rheumatology”, as well as using the literature on basic and clinical endocrinology, cardiology and pharmacology. The work was written using a systems approach, methods of analysis, induction and observation. Results and discussion. Systemic cortico- steroids are used in cases of severe and critical course of the disease COVID-19 (confirmation may be an increase in ferritin, procalcitonin, C-reactive protein (CRP), decreased cognitive functions, development of sopor). Their appointment is also justified when the initial course of the disease was not diagnosed as severe, but suddenly the patient’s condition deteriorated. The use of antibiotic therapy is advisable when a bacterial infection is attached - (procalcitonin (PCT)> 0.5 ng / ml, purulent sputum, leukocytosis> 12 × 109 / L (in the absence of previous use of glucocorticoids), an increase in band neutrophils of more than 10%). In the presence of chronic infectious diseases in patients with COVID-19 (for ex- ample, chronic obstructive pulmonary disease-COPD, chronic pyelonephritis, etc.), antibiotics are prescribed to prevent exacerbations of these diseases. Conclusion. In the course of the study, the authors of the article formulated the follow- ing principles of glucocorticoid therapy: drugs should be prescribed according to strict indications; maximum doses are applied in a short course; when the patient’s condition is stabilized, it is necessary to reduce the dose in a timely manner and gradually to complete withdrawal to prevent the development of “withdrawal” syndrome, adrenal insufficiency of cen- tral genesis, sympathoadrenal crises; during and after treatment, prevention of complications of glucocorticoid therapy (hyperglycemia, hypocalcemia, osteopenia, inflammatory diseases of the urinary system) is recommended; collegial management of patients by infectious diseases and endocrinologists is mandatory.

Staphylococcus aureus-induced pyogranulomatous dermatitis, osteomyelitis, and meningitis with Splendore-Hoeppli reaction in a cat coinfected with the feline leukemia virus and Leishmania sp.

This report describes a case of a pyogranulomatous dermatitis, osteomyelitis, and meningitis with Splendore-Hoeppli reaction caused by Staphylococcus aureus in a sheltered female adult cat coinfected with the feline leukemia virus (FeLV) and Leishmania sp. The cat had a mild anemia and marked increased total leukocytes, particularly band and segmented neutrophils. The cat had laboratorial diagnosis of FeLV and Leishmania sp. infections. Clinically, the cat had extensive and multifocal areas of ulceration in cranial region. Due to the progression of cutaneous lesions, progressive weight loss, and the risk for other sheltered animals, the cat was euthanized. Microscopically, there was marked pyogranulomatous ulcerative dermatitis, osteomyelitis and meningitis, with multiple large intralesional colonies of Gram-positive cocci associated with Splendore-Hoeppli reaction. Aerobic bacterial isolates were identified as S. aureus by MALDI-ToF MS. Leishmania sp. DNA sequences were detected in liver and spleen, and amastigotes were demonstrated in skin sections by immunohistochemistry. In conclusion, here we describe a case of S. aureus-induced pyogranulomatous meningitis with SH reaction in a cat naturally coinfected with FeLV and Leishmania sp.

Describing Characteristics and Differences of Neutrophils in Sepsis, Trauma, and Control Patients in Routinely Measured Hematology Data.

Neutrophils have an important role in the immune response. These cells can be subjected to an impaired function and a shift in population depending on disease states. In sepsis, this shift is recognized and flagged by automated hematology analyzers, including the presence of band neutrophils, while these cells, although present, appear not to be detected in trauma patients. To better understand this suspected error in flagging, we set out to distinguish neutrophil populations of these two patient groups and compared these with controls. Different data-driven methods were used compared to standard algorithms used by the software of the analyzers. Using K-means clustering, we extracted neutrophils from raw hematology analyzer datafiles, and compared characteristics of these clusters between the patient groups. We observed an increased neutrophil size for both sepsis and trauma patients, but trauma patients had a smaller increase. Trauma patients also had a high proportion of cells with relatively high nuclear segmentation, which is contradictory with the presence of band neutrophils. This, in combination with the smaller size increase, might explain the inability to flag band neutrophils in trauma.

Severe COVID-19 Shares a Common Neutrophil Activation Signature with Other Acute Inflammatory States.

Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaki disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflammatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at the protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.