Leukocyte Integrin CD18 Expression Mediates Transient Neutropenia Following G-CSF Administration.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3587-3587
Author(s):  
Laura Tuschong ◽  
Catherine E. Dejesus ◽  
Meredith Adams ◽  
Aylin C. Bonifacino ◽  
Dennis D. Hickstein ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Cell Counts ◽  
Transient Loss ◽  
The Mean ◽  
White Blood Cell Counts

Abstract Abstract 3587 Poster Board III-524 The mechanism whereby neutrophils traffic from the circulation in response to G-CSF has remained unclear despite the observation of ourselves and others that there is a dramatic, yet transient, loss of circulating neutrophils shortly following the administration of G-CSF in humans, non-human primates, and mice (Gordon BC, et al. Exp Hematol. 35:872-8, 2007). To determine the role of the CD18 leukocyte integrin on neutrophils in the egress of neutrophils from the circulation, we used dogs with canine leukocyte adhesion deficiency (CLAD), a genetic disease in which a mutation in CD18 prevents CD18 surface expression. We selected CLAD dogs who had 5-10% CD18+ neutrophils following either matched littermate allogeneic transplant or autologous gene therapy for CLAD. Three CLAD dogs meeting these criteria were evaluated. Three carrier dogs served as controls. G-CSF was administered at 10μg/kg SQ to all six animals. Peripheral blood samples (EDTA) were taken immediately prior to G-CSF administration, and at 15, 30, 60, 120, 240 minutes, and 24 hours following G-CSF administration. Total white blood cell counts, neutrophil counts, and the number and percentage of CD18+ peripheral blood leukocytes were assessed. As anticipated, the control dogs had a 60% decrease in circulating neutrophils 30 minutes following G-CSF administration: the mean +/− standard of deviation (SD) absolute neutrophil baseline count decreased from 6806+/−1072/μL to 2727+/−767/μL. In five control animals the neutrophil nadir occurred at 30 minutes post-G-CSF, and in one control dogs it occurred 15 minutes following G-CSF administration. Experimental CLAD dogs had only a 35% decline in neutrophil numbers at 30 minutes, from a mean baseline of 6777+/− 672/μL to 4433+/−265/μL. In these dogs the neutrophil count returned to pre-G-CSF levels by 60 minutes post-G-CSF. By 24 hours after G-CSF, the neutrophil level was increased 3-fold from baseline. Immunophenotyping using an anti-CD18 and a canine specific anti-neutrophil PE conjugated antibody indicated that only the CD18+ neutrophils disappeared from the circulation following G-CSF administration. At baseline the transplanted CLAD dogs had a mean of 15.2+/−3.9% CD18+ peripheral blood leukocytes, of these 50.7+/−7.1% were CD18+ neutrophils. Thirty minutes following G-CSF administration the mean+/−SD percentage of CD18+ leukocytes declined to 13.7+/−3.7% with 33.4+/−5.8% being neutrophils. There was also a slight decline in CD14+CD18+ monocytes from 6.2 +/− 1.5% to 4.0 +/− 1.2%, which was not observed in the controls. There was no change in CD18- leukocyte numbers. The percentage of CD18+ neutrophils returned to baseline by 60 minutes and remained there at subsequent time points. These results demonstrate that the CD18 leukocyte integrin on circulating neutrophils mediates the transient neutropenia associated with G-CSF administration. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hyperexpression of TLR2 and TLR4 in patients with ischemic stroke in acute period of the disease

2020 ◽  
Vol 22 (4) ◽  
pp. 665-674
Author(s):  
L. V. Gankovskaya ◽  
L. V. Stakhovskaya ◽  
V. V. Grechenko ◽  
E. A. Koltsova ◽  
O. S. Uvarova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Innate Immunity ◽  
Ischemic Stroke ◽  
Peripheral Blood ◽  
Surface Expression ◽  
Control Group ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Healthy Donors ◽  
Tlr4 Gene

Pathogenesis of ischemic stroke  is actively  involved  in the  system  of innate immunity. Under conditions of cerebral  ischemia, a number of biologically  active  substances are  released  that  interact with innate immunity receptors, in particular TLR2  and  TLR4, which  exacerbate inflammation in brain  tissue. Identification of predictor markers  at the level of the innate immunity system may foresee the clinical course of ischemic stroke and ensure timely treatment. Our objective was to study expression of TLR2 and TLR4 receptors in peripheral blood leukocytes  in patients with ischemic stroke in the dynamics of the disease. 27 people  were included in the study. The main  group consisted of patients with ischemic stroke of varying severity (n = 19). Patients of the main  group were divided into two subgroups:  with an NIHSS index value of < 10 (n = 10) and > 10 (n = 9). The control group included healthy  donors  with no history  of acute  and chronic inflammatory diseases (n = 8). Peripheral blood  leukocytes  were used as the  test material. To determine expression  of the TLR2  and TLR4  genes, RT-PCR in real time was used. Surface  expression  of TLRs was determined by flow cytometry. A study of the TLR2 and TLR4 gene expression showed that on the 1st, 3rd  and 7th  day post-stroke, the TLR4 gene expression  in patients was significantly  increased, when compared to the control group (p < 0.01), whereas TLR2 gene expression on the 3rd  day of the disease was not statistically different from the control group. A study of surface expression  of receptors showed that the average TLR2 fluorescence intensity on the patients’ peripheral blood monocytes was significantly  increased on the 1st  and 3rd  day of disease when compared to the control group.  The  surface  expression  of TLR4  on monocytes has a statistically significant  increase  only on day 7. Assessment  of surface expression  of TLRs in subgroups  with different  severity values by NIHSS showed that  patients with a NIHSS index > 10 had a significantly  higher  level of surface of TLR2  expression  over the observation period, while the largest difference in TLR4  expression  in the subgroups  was observed  on the 1st day of the disease (p < 0.05). Patients with ischemic stroke showed an increase  in TLR2 and TLR4 expression at the gene and protein level, compared to healthy  donors. These indices can be considered possible predictors for clinical  prognosis  of ischemic stroke.

Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1268-1277 ◽  
Author(s):  
M Carbonari ◽  
M Cibati ◽  
M Cherchi ◽  
D Sbarigia ◽  
AM Pesce ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Hiv Infection ◽  
Peripheral Blood ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Cell Counts ◽  
Immunodeficiency Virus

We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

scholarly journals Increase Expression of the Genes PADI4, MPO and ELANE in the Chronic Phase of DVT Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4921-4921
Author(s):  
Isabella Macedo Toni ◽  
Kiara Zapponi ◽  
Bidossessi Wilfried Hounkpe ◽  
Beatriz Moraes Martinelli ◽  
Silmara Aparecida De Lima Montalvão ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Rna Extraction ◽  
Chronic Phase ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Membrane Rupture

Venous thromboembolism (VTE) is a common multifactorial disease, and the knowledge in the pathophysiology can improve prophylaxis and treatment. The myeloid lineage, especially neutrophils, have been implied in the pathophysiology of VTE, but it is still unclear whether its activation is persistent after the acute phase of the disease. In addition to phagocytosis, neutrophils participate of the innate immunity through the release of NETs (Neutrophil Extracellular Traps): Reactive Oxygen Species (ROS)-triggered antimicrobial structures composed of DNA lined with granular components (Brinkmann et al., 2004). NETs also contribute to the coagulation, as they facilitate the interaction between erythrocytes, leukocytes, platelets and endothelium, besides potentially inhibit anticoagulant pathways (Fuchs et al., 2012). The molecular mechanisms underlying the "NETosis" are still being characterized, but it is known that following inflammatory stimuli, the generation of ROS leads to the dissolution of intracellular membranes and translocation of Myeloperoxidase (MPO) and Neutrophil Elastase (ELANE) to nucleus. Histones are then citrullinated by Peptidyl Arginine Deiminase 4 (PADI4) and degraded by ELANE, promoting chromatin decondensation, plasma membrane rupture and NET's release. Previously we showed increased neutrophil activation, adhesive properties and the presence of NETs serum markers in patients with VTE in the chronic phase (Zapponi et al., 2014; Zapponi, manuscript in preparation). However, assessment of NETs activity is challenging and, until now, there is no consensus in markers or gold standards measurements. We decided to investigate if the expression of the genes PADI4, ELANE, MPO, could provide additional information regarding NETosis activity, through an indirect analysis of the process by Real-Time PCR. We analyzed samples from 20 controls and 19 patients previously included in the NET study, respecting the time window in which the NETs plasma markers were assessed (between 3 and 43 months after the TVP event). RNA extraction was performed from peripheral blood leukocytes using Trizol reagent, according to the manufacturer's protocol. Real time-PCR reactions were performed with qPCRBIO SyGreen mix (PCRBio), on RotorGene Q (Qiagen) with a reaction efficiency above 0.99. The relative fold changes (Fc) of the genes PADI4, ELANE, MPO were calculated using the Pfaffl method (Pfaffl, 2001 - Nucleic Acids Res.) and the significance level of Mann-Whitney test was considered if p <0.05. The 39 cDNA samples were amplified in duplicates and a standard sample was included for normalization between different runs. The threshold values (Ct) from PADI4, ELANE, MPO were adjusted for the individual's neutrophil proportion and the geometric means of the housekeeping genes (Actin Beta and Glyceraldehyde-3-Phosphate Dehydrogenase) were used for normalization between samples. The standard deviation was 0.30 between runs and ranged between duplicates from 0 to 1.16 (0.096 on average). The patients showed significant increased expression of PADI4 (p <0.01), MPO and ELANE (p <0.05) when compared to controls (Figure 1). Neutrophils emerges fully differentiated from the bone marrow and ranges from 40 to 70% of circulating leukocytes (Cowland and Borregaard, 2016). Isolating these cells can be challenging and the process itself may influence expression profile. The use of total leukocytes would not be the most appropriate process but considering that the analyzed genes are predominantly expressed in neutrophils, the Ct values were weighted according to the neutrophil to leucocytes proportions of each individual. Our results indicate that the increased neutrophils activation in patients can be represented in PADI4, ELANE and MPO gene expression of peripheral blood leukocytes, which could better represent NETotic activity in these patients. We cannot rule out that the expression of these genes in myeloid precursors could better represent NETotic activity, but our results indicate that this analysis may also be viable in peripheral blood. Disclosures No relevant conflicts of interest to declare.

Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1241-1241
Author(s):  
Mark C Lanasa ◽  
Sallie D Allgood ◽  
Susan L Slager ◽  
Nicola J Camp ◽  
Neil E. Kay ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Surface Expression ◽  
Cellular Activation ◽  
Immunoglobulin Isotype ◽  
Cell Counts ◽  
Intracellular Expression ◽  
Trisomy 12

Abstract Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1268-1277 ◽  
Author(s):  
M Carbonari ◽  
M Cibati ◽  
M Cherchi ◽  
D Sbarigia ◽  
AM Pesce ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Hiv Infection ◽  
Peripheral Blood ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Cell Counts ◽  
Immunodeficiency Virus

Abstract We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

scholarly journals Enzymatic Activities of Bovine Peripheral Blood Leukocytes and Milk Polymorphonuclear Neutrophils during Intramammary Inflammation Caused by Lipopolysaccharide

2002 ◽  
Vol 9 (4) ◽  
pp. 812-817 ◽  
Author(s):  
C. Prin-Mathieu ◽  
Y. Le Roux ◽  
G. C. Faure ◽  
F. Laurent ◽  
M. C. Béné ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Clinical Signs ◽  
Enzymatic Activities ◽  
Polymorphonuclear Neutrophils ◽  
Live Cells ◽  
Mammary Tissue ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Cell Counts

ABSTRACT Leukocytes are recruited from peripheral blood into milk as part of the inflammatory response to mastitis. However, excessive accumulation of inflammatory cells alters the quality of milk and the proteases produced by polymorphonuclear neutrophils (PMNs) and macrophages may lead to mammary tissue damage. To investigate PMN recruitment and the kinetics of their intracytoplasmic enzymes in inflammation, we generated mastitis in six cows by intramammary infusion of lipopolysaccharide (LPS). Clinical signs of acute mastitis were observed in all of the cows, and normal status was resumed by 316 h. Intracytoplasmic elastase, collagenase, and cathepsin activities were measured within live cells by flow cytometry in peripheral blood leukocytes and milk PMNs before and during the inflammatory process (at 10 time points between 4 and 316 h). The proportion of immature PMNs was appreciated by CD33 surface labeling measured in flow cytometry. Leukopenia was observed in the peripheral blood 4 h postinfusion, concomitant to an increase in somatic cell counts in milk. CD33+ PMNs were preferentially recruited from the peripheral blood to milk. Enzymatic activities were detected in PMNs, lymphocytes, and monocytes at levels depending on the cell type, sample nature, and time of collection. Milk PMNs had lower enzymatic activities than peripheral blood PMNs. This study showed that milk PMNs recruited during LPS-induced experimental mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs. This suggests that CD33, an adhesion molecule, may be involved in the egress from blood to milk and that the enzymatic contents of PMNs are partly used during this process.

scholarly journals Longitudinal Transcriptional Analysis of Peripheral Blood Leukocytes in COVID Convalescent Donors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1767-1767
Author(s):  
Mallikarjuna Rao Gedda ◽  
Patrick Danaher ◽  
Lipei Shao ◽  
Martin Ongkeko ◽  
Leonard Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Peripheral Blood ◽  
Interferon Signaling ◽  
Peripheral Blood Leukocytes ◽  
Active Infection ◽  
Blood Leukocytes ◽  
Chemokine Signaling ◽  
The Mean ◽  
Type Iii Interferon

Abstract Abstract Introduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) can induce a strong host immune response. Several groups have investigated the course of antibody responses in patients recovering from SARS-CoV-2 infections but little is known about the recovery of cellular immunity. This study investigated the cellular immune response in people who had recovered from SARS-CoV2 infection. Methods 162 coronavirus disease 2019 (COVID-19) convalescent plasma donors (CCD) and 40 healthy donor (HD) controls were enrolled prospectively in an IRB-approved protocol (Clinical Trials Number: NCT04360278) and provided written informed consent to participate in the study. Using the nCounter platform and host response panel with 785 genes across more than 50 pathways, we compared transcriptomic profiles on RNA samples obtained from the peripheral blood leukocytes of these 162 CCD and 40 HD. Additionally, in 69 of the 162 CCD samples, we evaluated transcriptomic trends at more than one-time point during the convalescent period. Results Age, sex, ethnicity, and body mass index distributions were similar among the CCD and HD. With respect to baseline complete blood counts, hemoglobin, platelets, and absolute basophil and eosinophil counts, all were similar among CCD and HD (Table 1). However, despite sample collections occurring several days after convalescence, mean counts for absolute neutrophil counts, absolute monocyte counts, and absolute lymphocyte counts were significantly higher among CCD compared to HD. 30-90 days after diagnosis, 19 of 773 genes differed (FDR &lt; 0.05) between the average CCD and HD samples. Up-regulated genes included MAFB, CTLA4, PTGS2, and the chemokine signaling genes CXCR4, CXCL5, CXCL2 and CCR4. Down-regulated genes included PTGER2, CASP8, and the interleukins IL36A, IL31, IL20 and IL21 (Figure 1 a,b). Differential gene expression persisted for months. At 90-120 days, 13 genes were differentially regulated, including again MAFB CXCR4, PTGS2, CXCL2 and PTGER2, plus SMAD4. At 120-150 days post-diagnosis, 58 genes were differentially expressed (FDR &lt; 0.05) compared to HD. Pathways with up-regulated genes included Treg differentiation, type III interferon signaling and chemokine signaling. 150-360 days post-diagnosis, 4 genes remained up-regulated on average (FDR &lt; 0.05): PTGS2, PIK3CR, CXCL1 and SMAD4 (Figure 1 c,d). Individual patients varied considerably from the mean trend. Scoring samples by their similarity to the gene expression profile of the mean HD sample, 21 CCD samples from 20 unique patients (12%) were identified as highly perturbed from HD. 84% of these highly perturbed samples were collected &gt; 90 days post-diagnosis. Of these 21 samples, 6 were distinguished by &gt; 2-fold up-regulation of a cluster of interleukin and type-1 interferon genes (Figure 2). Conclusions Overall, our study identified important gene expression trends in CCD compared to HD in the post-acute period. The changes varied with time and among donors. As the expression of T-cell inhibitory molecule CTLA4 fell, the number of differentially expressed increased with the most marked changes occurring 120 to 150 days post-diagnosis in genes in chemokine signaling, type III interferon signaling and Treg pathways. Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed in individuals following recovery from COVID-19 infection. Our data may serve as the basis for risk modification strategies in the period of active infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. Figure 1 Figure 1. Disclosures Danaher: NanoString Technologies: Current Employment, Current holder of individual stocks in a privately-held company.

ENDOTHELIAL-DEPENDENT MECHANISMS OF LEUKOCYTE ADHESION: ROLE OF MONOKINES

1987 ◽  
Author(s):  
M A Gimbrone ◽  
M P Bevilacqua ◽  
M E Wheeler
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Cell ◽  
Surface Structure ◽  
Peripheral Blood ◽  
Leukocyte Adhesion ◽  
Active Role ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes

Localized adhesion of peripheral blood leukocytes to the vessel wall is an essential component of inflammatory reactions. There is increasing experimental evidence that vascular endothelial cells play an active role in this process. Our laboratory has been especially interested in defining endothelial-dependent mechanisms of leukocyte adhesion, and the role of leukocyte products in their modulation. We have reported1 that purified natural human monocyte-derived interleukin 1 (IL-1) can act directly on cultured human endothelial cells (HEC) to dramatically increase the adhesiveness of their surfaces for human polymorphonuclear leukocytes (PMN), monocytes and the related cell lines HL-60 and U937. This effect was concentration-, time- (onset≅30 min; peak≅4h) , and protein/RNA-synthesis-requiring, and, in selective pretreatment/fixation experiments, was shown to be mediated primarily through the endothelial cell. To better define this inducible endothelial pro-adhesive mechanism, we have developed a series of murine monoclonal antibodies directed against monokine-stimulated HEC surfaces. One of these antibodies (H4/18) recognizes an endothelial cell surface structure which is induced by IL-1 (and certain other cytokines)2 in a similar fashion (kinetics, concentration - dependence, sensitivity to metabolic inhibitors) as the pro-adhesive surface change for leukocytes. H4/18 partially blocks HD-60 cell adhesion to monokine-treated HEC, and, in vivo, labels human vascular endothelium at sites of experimental delayed hypersensitivity reactions4. A second monoclonal antibody (H18/7)5 significantly blocks the adhesion of both HL-60 cells and PMN to monokine-treated HEC. Monoclonal antibodies H4/18 and H18/7 appear to recognize the same inducible surface structure as assessed by immunoprecipitation of extracts of metabolically labeled, monokine-stimulated HEC. We have designated this monokine-inducible, endothelial-leukocyte adhesion molecule "E-IAM 1". IL-1 treated HEC cultures (in contrast to sham-treated control cultures) generate a soluble leukocyte adhesion inhibitor (LAI)6,7. LAI acts on PMN to inhibit their adhesion to hyperadhesive endothelial monolayers as well as to serum-coated plastic surfaces, but does not inhibit PMN activation by chemotactic stimuli (LTB4, f-met-leu-phe). IAI appears to differentially inhibit adhesion of peripheral blood leukocytes, isolated from the same donor, to hyperadhesive HEC (PMN > monocytes; lymphocytes, no effect), and does not inhibit HL-60 cell-HEC adhesion. Endothelial production of IAI is time-dependent (peak 5-6 h.), and blocked by cycloheximide but not by aspirin. Preliminary characterization indicates that LAI is nonsedimentable (250,000 xg, 45 min), nondialyzable (>10 kD), stable to heat (80°C, 30 min) and acid (pH 2) and is precipitable by ammonium sulphate (60-80% saturation). Thus, this endothelial-derived inhibitory activity, which appears to be distinct from PGI2 or other cyclooxygenase products, blocks leukocyte adhesion without globally suppressing leukocyte function. Further characterization of the cellular and molecular mechanisms regulating the endothelial expression of E-LAM 1 and LAI should contribute to our understanding of the active role of the vascular wall in the inflammatory process.1. Bevilacqua et al. (1985); J. Clin. Invest.76:2003.2. Cotran et al. (1986); J. Exp. Med. 164:661.3. Bevilacqua et al. (1987); Fed. Proc. (in press).4. Wheeler et al. (1986); Fed. Proc. 45:1725.5. Wheeler et al. (1987); Fed. Proc. (in press).

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