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Signal Transduction and Targeted Therapy May 2021Severely immunosuppressed AIDS patients with recurrent opportunistic infections (OIs) represent an unmet medical need even in the era of antiretroviral therapy (ART).... (Clinical Trial)
Clinical Trial
Severely immunosuppressed AIDS patients with recurrent opportunistic infections (OIs) represent an unmet medical need even in the era of antiretroviral therapy (ART). Here we report the development of a human leukocyte antigen (HLA)-mismatched allogeneic adaptive immune therapy (AAIT) for severely immunosuppressed AIDS patients. Twelve severely immunosuppressed AIDS patients with severe OIs were enrolled in this single-arm study. Qualified donors received subcutaneous recombinant granulocyte-colony-stimulating factor twice daily for 4-5 days to stimulate hematopoiesis. Peripheral blood mononuclear cells were collected from these donors via leukapheresis and transfused into the coupled patients. Clinical, immunological, and virological parameters were monitored during a 12-month follow-up period. We found AAIT combined with ART was safe and well-tolerated at the examined doses and transfusion regimen in all 12 patients. Improvements in clinical symptoms were evident throughout the study period. All patients exhibited a steady increase of peripheral CD4 T cells from a median 10.5 to 207.5 cells/μl. Rapid increase in peripheral CD8 T-cell count from a median 416.5 to 1206.5 cells/μl was found in the first 90 days since initiation of AAIT. In addition, their inflammatory cytokine levels and HIV RNA viral load decreased. A short-term microchimerism with donor cells was found. There were no adverse events associated with graft-versus-host disease throughout the study period. Overall, AAIT treatment was safe, and might help severely immunosuppressed AIDS patients to achieve a better immune restoration. A further clinical trial with control is necessary to confirm the efficacy of AAIT medication.
Topics: Acquired Immunodeficiency Syndrome; Adolescent; Adoptive Transfer; Adult; Allografts; Female; HIV-1; HLA Antigens; Humans; Leukocytes, Mononuclear; Male; Middle Aged
PubMed: 33958574
DOI: 10.1038/s41392-021-00550-2 -
STAR Protocols Dec 2021Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent...
Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).
Topics: Computational Biology; Flow Cytometry; Humans; Leukocytes, Mononuclear; Monitoring, Immunologic; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 34806044
DOI: 10.1016/j.xpro.2021.100900 -
Journal of Dairy Science Mar 1988In vitro effects of killed Staphylococcus aureus cells on bovine blood mononuclear leukocytes from uninfected cows or cows with chronic staphylococcal mastitis were...
In vitro effects of killed Staphylococcus aureus cells on bovine blood mononuclear leukocytes from uninfected cows or cows with chronic staphylococcal mastitis were assessed using a lymphocyte proliferation assay and a [51Cr] release cytotoxicity assay. Killed S. aureus cells cultured with mononuclear leukocytes caused a concentration-dependent decrease in lymphocyte proliferation that was associated with a concomitant decrease in mononuclear leukocyte viability. Responses of mononuclear leukocytes from uninfected and infected cows to killed S. aureus were similar, indicating effects were independent of the infection status of the animal. Addition of blood polymorphonuclear leukocytes to blood mononuclear leukocyte cultures without S. aureus cells did not alter mononuclear leukocyte viability but suppressed lymphocyte proliferation at the highest polymorphonuclear leukocyte:mononuclear leukocyte ratios (4:1 and 8:1) tested. When S. aureus cells and polymorphonuclear leukocytes were cultured with mononuclear leukocytes, both blood and milk polymorphonuclear leukocytes protected against the loss of viability compared with leukocytes cultured with S. aureus cells alone but did not consistently restore proliferative responses of the lymphocytes. These observations demonstrate that lymphocyte proliferation and mononuclear leukocyte viability are detrimentally affected by S. aureus cells, an effect that can be modulated by blood or milk polymorphonuclear leukocytes.
Topics: Animals; Cattle; Female; In Vitro Techniques; Leukocytes, Mononuclear; Lymphocyte Activation; Mastitis, Bovine; Neutrophils; Staphylococcal Infections; Staphylococcus aureus
PubMed: 3372823
DOI: 10.3168/jds.S0022-0302(88)79624-1 -
European Journal of Pharmacology Oct 2015Enhanced leukocyte recruitment is an inflammatory process that occurs during early phases of the vascular dysfunction that characterises atherosclerosis. We evaluated...
Enhanced leukocyte recruitment is an inflammatory process that occurs during early phases of the vascular dysfunction that characterises atherosclerosis. We evaluated the impact of anti-TNF-α (adalimumab, infliximab and etanercept) and anti-IL-12/23 (ustekinumab) on interactions between human leukocytes and endothelial cells in a flow chamber that reproduced in vivo conditions. Clinical concentrations of anti-TNF-α were evaluated on the leukocyte recruitment induced by a variety of endothelial (TNF-α, interleukin-1β, lymphotoxin-α and angiotensin-II) and leukocyte (PAF, IL-12 and IL-23) stimuli related to inflammation and atherosclerosis. Treatment with anti-TNF-α, even before or after establishing the inflammatory situation induced by TNF-α, diminished leukocyte-endothelial cell interactions induced by this stimuli. Our results also implicated adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in the actions of anti-TNF-α in terms of leukocyte adhesion to endothelium. However, anti-TNF-α drugs did not influence the actions of interleukin-1β, but prevented those of lymphotoxin-α and angiotensin-II. However, once established, inflammatory response elicited by the latter three stimuli could not be reversed. Pre-treatment with anti-TNF-α, also prevented leukocyte actions induced by IL-23 on PBMC rolling flux and rolling velocity and by IL-12 on PMN adhesion. Ustekinumab exhibited a more discreet profile, having no effect on leukocyte recruitment induced by any of the endothelial stimuli, while blocking the effects of IL-23 on leukocyte activation and those of IL-12 on PMN adhesion and PAF on PBMC rolling velocity. These findings endorse the idea that biological anti-inflammatory drugs, in particular anti-TNF-α, have the capacity to influence cardiovascular risk accompanying psoriasis and rheumatoid arthritis by ameliorating vascular inflammation.
Topics: Adalimumab; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-12; Interleukin-23; Leukocytes, Mononuclear; Tumor Necrosis Factor-alpha
PubMed: 26344475
DOI: 10.1016/j.ejphar.2015.08.054 -
Journal of Clinical Laboratory Analysis 2003In the general population, about 5% of individuals are homozygotic and 35% are heterozygotic carriers for chitotriosidase (ChT) deficiency. Activated macrophages are...
In the general population, about 5% of individuals are homozygotic and 35% are heterozygotic carriers for chitotriosidase (ChT) deficiency. Activated macrophages are considered to be the main source of plasma ChT activity, which permits the biochemical characterization of homozygote deficients. However, in the case of detecting heterozygotic carriers, the results are often inconclusive. The activities of ChT in plasma and mononuclear (MN) and polymorphonuclear (PMN) leukocytes were determined in 169 control subjects (72 males and 97 females) with a mean age (+/- SD) of 47.5+/-9.7 years (range 18-96 years). The specific enzyme activity was in PMN leukocytes >MN leukocytes >plasma, with a highly significant partial correlation being found between the activities of ChT in plasma and PMN leukocytes (r=0.578, P<0.001). A significant correlation was found between the age of the patients studied and plasma ChT activity (r=0.568, P<0.001). No significant correlation was found for enzyme activities in MN (r=0.105) or in PMN leukocytes (r=0.043). The results obtained suggest that, in normal physiological conditions, PMN leukocytes may secrete ChT to the plasma. Although the activities of ChT in MN and PMN leukocytes are not affected by demographic factors, it is not possible to use them for the biochemical detection of ChT-deficient heterozygotic carriers.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Hexosaminidases; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Neutrophils; Reference Values
PubMed: 14614752
DOI: 10.1002/jcla.10108 -
BMC Immunology Dec 2017To investigate natural killer (NK) cell activity, circulating cytokine level and peripheral blood mononuclear cell (PBMC) cytokine production status in critically ill...
BACKGROUND
To investigate natural killer (NK) cell activity, circulating cytokine level and peripheral blood mononuclear cell (PBMC) cytokine production status in critically ill patients.
METHODS
Blood samples were collected <24 h after admission from 24 intensive care unit (ICU) patients and 24 age-, sex-, and body mass index (BMI)-matched healthy controls. Serum cytokine concentrations and cytokine production by PBMCs and lipopolysaccharide (LPS)-stimulated PBMCs were measured.
RESULTS
The ICU group showed lower NK cell activity than the controls under all conditions and an absence of interferon (IFN)-γ. After adjusting for triglycerides, LDL- and HDL-cholesterol, and glucose, the ICU group exhibited lower serum levels of albumin and interleukin (IL)-12 and higher leukocyte counts and hs-CRP and IL-6 levels than the controls. Non-stimulated PBMCs from ICU patients secreted significantly greater amounts of IL-6 and IL-1β than the controls; however, the production of IL-6, TNF-α and IL-1β in response to LPS stimulation was significantly lower in the ICU group.
CONCLUSIONS
Significant reductions in NK cell activity and serum IL-12 level, an absence of serum IFN-γ, and decreased cytokine production from LPS-stimulated PBMCs indicate the hyporesponsiveness of NK cells and an impaired early phase inflammatory response in critically ill patients (ClinicalTrials.gov NCT02565589 :). Retrospectively registered; October 1, 2015.
Topics: Adjuvants, Immunologic; Aged; Case-Control Studies; Critical Illness; Cytokines; Female; Humans; Inflammation; Intensive Care Units; Killer Cells, Natural; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Middle Aged
PubMed: 29221433
DOI: 10.1186/s12865-017-0231-y -
American Journal of Physiology. Cell... Dec 2009Gastric mucosal inflammation is frequently associated with hypergastrinemia, and a correlation exists between the level of gastrin and degree of gastritis. We have...
Gastric mucosal inflammation is frequently associated with hypergastrinemia, and a correlation exists between the level of gastrin and degree of gastritis. We have previously observed that gastrin promotes leukocyte-endothelial interactions and contributes to Helicobacter-induced inflammation in the rat mesentery. In the present study, we aimed to evaluate a possible proinflammatory activity of gastrin in humans. The interaction between human leukocytes [U-937 cells, peripheral blood polymorphonuclear (PMN), and peripheral blood mononuclear (PBMC) cells] and human umbilical vein endothelial cells (HUVEC) was analyzed in static and dynamic conditions. The endothelial expression of adhesion molecules [P-selectin, E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1] was analyzed by flow cytometry and fluorescent microscopy screening. Gastrin increased the static adhesion of U-937 cells to HUVEC (1 h; 10(-9) M: 122 +/- 9%; 10(-8) M: 143 +/- 17%; 10(-7) M: 162 +/- 14% vs. control, all P < 0.05). Incubation of HUVEC with gastrin (4 h) also increased PBMC rolling (vehicle: 63 +/- 12; 10(-9) M: 109 +/- 29; 10(-8) M: 141 +/- 24; 10(-7) M: 261 +/- 16 leukocytes/min, P < 0.05) and adhesion (vehicle: 3 +/- 2, 10(-9) M: 11 +/- 4, 10(-8) M: 17 +/- 5, 10(-7) M: 15 +/- 5 leukocytes/mm(2), all P < 0.05) in the parallel-plate flow chamber. Treatment of PBMC with gastrin had no effects. The cholecystokinin (CCK)-2 receptor antagonist (L-365,260, 10(-7) M) prevented the effects of gastrin. P-selectin and VCAM-1 expression were enhanced by gastrin, and neutralizing antibodies against these molecules prevented PBMC rolling and adhesion. Gastrin did not affect the interactions between HUVEC and PMN. Gastrin induces interactions between human mononuclear leukocytes and endothelial cells through the activation of CCK-2 receptors and the enhancement of endothelial P-selectin and VCAM-1.
Topics: Cell Adhesion; Cell Communication; Cells, Cultured; Endothelial Cells; Flow Cytometry; Gastrins; Humans; Leukocyte Rolling; Leukocytes, Mononuclear; Microscopy, Fluorescence; P-Selectin; Receptor, Cholecystokinin B; Umbilical Veins; Vascular Cell Adhesion Molecule-1
PubMed: 19812370
DOI: 10.1152/ajpcell.00082.2009 -
Frontiers in Endocrinology 2022Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are... (Review)
Review
Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8 T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop β-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of expanded CD8 T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the β-cell/immune crosstalk in an islet microenvironment; the features that make β-cells more sensitive to autoimmunity; therapeutic agents that may modulate β-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor.
Topics: Humans; Diabetes Mellitus, Type 1; CD8-Positive T-Lymphocytes; Leukocytes, Mononuclear; Pluripotent Stem Cells; Cell Death
PubMed: 36726462
DOI: 10.3389/fendo.2022.1076683 -
Journal of Translational Medicine May 2019Rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. Our previous study revealed a downregulation of genes...
BACKGROUND
Rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. Our previous study revealed a downregulation of genes related to T lymphocytes and an upregulation of genes related to monocytes and neutrophils after IA rupture. It remains unknown whether that resulted from alterations in transcription or cell count. We sought to characterize the systemic response to IA rupture through analysis of transcript expression profiles in peripheral blood cells. We also investigated effects of IA rupture on the composition of mononuclear cells in peripheral blood.
METHODS
We included 19 patients in the acute phase of IA rupture (RAA, first 72 h), 20 patients in the chronic phase (RAC, 3-15 months), and 20 controls. Using deep transcriptome sequencing, we analyzed the expression of protein-coding and noncoding RNAs. Expression levels, transcript biotypes, alternative splicing and other features of the regulated transcripts were studied. A functional analysis was performed to determine overrepresented ontological groups among gene expression profiles. Flow cytometry was used to analyze alterations in the level of mononuclear leukocyte subpopulations.
RESULTS
Comparing RAA and controls, we identified 491 differentially expressed transcripts (303 were downregulated, and 188 were upregulated in RAA). The results indicate that the molecular changes in response to IA rupture occur at the level of individual transcripts. Functional analysis revealed that the most impacted biological processes are related to regulation of lymphocyte activation and toll-like receptor signaling pathway. Differences between RAC and controls were less prominent. Analysis of leukocyte subsets revealed a significantly decreased number of CD4+ lymphocytes and increase of classical and intermediate monocytes in RAA patients compared to controls.
CONCLUSIONS
IA rupture in the acute phase strongly influences the transcription profiles of peripheral blood cells as well as the composition of mononuclear cells. A specific pattern of gene expression alteration was found, suggesting a depression of lymphocyte response and enhancement of monocyte activity.
Topics: Aneurysm, Ruptured; Female; Gene Expression Regulation; Humans; Intracranial Aneurysm; Leukocytes, Mononuclear; Male; Middle Aged; Molecular Sequence Annotation; Protein Isoforms; RNA, Messenger; Reproducibility of Results; Transcription, Genetic; Transcriptome
PubMed: 31046777
DOI: 10.1186/s12967-019-1891-6 -
Analytical Chemistry Sep 2018Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was...
Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was developed for mass cytometric analysis. By using 3 metal isotopes and 4 ratio intensity levels, 16 barcodes were generated to code, and later decode, cell samples in mass cytometry. The ratiometric Ln-Pdot barcodes not only provided high-mass-signal intensities but also eliminated the bias caused by different concentrations of the labeling reagents/barcodes and run-to-run differences in cell labeling efficiency. The ability to distinguish clearly the 16 sets of labeled MCF-7 cells with mass cytometry demonstrated the excellent resolving power of the ratiometric Ln-Pdot barcodes. Furthermore, the results from barcoding PBMC samples via CD45-specific cellular targeting indicated that the ratiometric Ln-Pdot barcodes could facilitate mass cytometry in high-throughput and multiplexed analysis, especially with precious human samples.
Topics: Cell Separation; Endocytosis; Flow Cytometry; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Leukocyte Common Antigens; Leukocytes, Mononuclear; MCF-7 Cells; Radiometry; Semiconductors
PubMed: 30139253
DOI: 10.1021/acs.analchem.8b03201