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The Journal of Clinical Investigation Feb 1980Factors influencing basophil production from the bone marrow of ovalbumin (OA)-sensitized guinea pigs have been examined in vitro. Autologous co-cultures of marrow and...
Factors influencing basophil production from the bone marrow of ovalbumin (OA)-sensitized guinea pigs have been examined in vitro. Autologous co-cultures of marrow and spleen cells from OA-immune animals contained significantly higher numbers of basophils after 7 d of liquid culture in the presence of OA, compared with control co-cultures or with marrow cultures alone (P < 0.005). Basophils increased in co-culture as the number of spleen cells added to a fixed number of marrow cells was increased from 0.10 to 2.5 x 10(6)/ml; at each spleen cell concentration, the presence of OA significantly enhanced basophil production in vitro when compared with unstimulated co-cultures. There was no basophil production from spleen cell suspensions cultured in the absence of autologous marrow cells. Conditioned media (CM) prepared from OA-stimulated spleen cells of OA-treated animals (CM-OA) caused a specific stimulation of basophil production from normal guinea pig bone marrow cells in liquid cultures (P < 0.01). Phytohemagglutinin (PHA)- and pokeweed mitogen-stimulated CM (CM-PHA, CM-pokeweed mitogen) nonspecifically enhanced normal basophilopoiesis, causing dose-dependent increases in basophils and histamine in vitro. CM-OA and CM-PHA also preferentially stimulated formation of neutrophil-macrophage colony-forming units in semisolid methylcellulose cultures.CM-PHA prepared from T cell-enriched splenic cell suspensions contained basophil-stimulating activity, whereas T cell-depleted CM-PHA activity did not exceed control values (P < 0.01). Preliminary characterization of CM-PHA revealed that basophil-stimulating activity was predominantly heat stable and nondialyzable. These results demonstrate OA-specific, as well as mitogen-dependent T-cell regulation of guinea pig basophilopoiesis in vitro. The data are compatible with the existence of a specific "basophilopoietin" in CM derived from guinea pig splenic T cells.
Topics: Animals; Basophils; Bone Marrow Cells; Cell Communication; Colony-Forming Units Assay; Culture Media; Guinea Pigs; Hematopoiesis; Histamine; In Vitro Techniques; Mitogens; Ovalbumin; Phytohemagglutinins; Spleen; T-Lymphocytes
PubMed: 6965379
DOI: 10.1172/JCI109682 -
Veterinary Immunology and... May 2020CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow...
CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than 90 % for both stained and unstained cells. Based on these data, CellTrace Violet™ is not recommended for the assessment of lymphocyte proliferation in equine cells. The mechanism of inhibition of equine lymphocyte proliferation by CellTrace Violet™ is unknown.
Topics: Animals; Cell Proliferation; Cell Survival; Concanavalin A; Flow Cytometry; Fluorescent Dyes; Horses; Lymphocyte Activation; Lymphocytes; Pokeweed Mitogens
PubMed: 32229340
DOI: 10.1016/j.vetimm.2020.110037 -
Frontiers in Immunology 2023The understanding of the pathophysiology of multiple sclerosis (MS) has evolved alongside the characterization of cytokines and chemokines in cerebrospinal fluid (CSF)...
INTRODUCTION
The understanding of the pathophysiology of multiple sclerosis (MS) has evolved alongside the characterization of cytokines and chemokines in cerebrospinal fluid (CSF) and serum. However, the complex interplay of pro- and anti-inflammatory cytokines and chemokines in different body fluids in people with MS (pwMS) and their association with disease progression is still not well understood and needs further investigation. Therefore, the aim of this study was to profile a total of 65 cytokines, chemokines, and related molecules in paired serum and CSF samples of pwMS at disease onset.
METHODS
Multiplex bead-based assays were performed and baseline routine laboratory diagnostics, magnetic resonance imaging (MRI), and clinical characteristics were assessed. Of 44 participants included, 40 had a relapsing-remitting disease course and four a primary progressive MS.
RESULTS
There were 29 cytokines and chemokines that were significantly higher in CSF and 15 in serum. Statistically significant associations with moderate effect sizes were found for 34 of 65 analytes with sex, age, CSF, and MRI parameters and disease progression.
DISCUSSION
In conclusion, this study provides data on the distribution of 65 different cytokines, chemokines, and related molecules in CSF and serum in newly diagnosed pwMS.
Topics: Humans; Cytokines; Multiple Sclerosis; Chemokines; Body Fluids; Disease Progression; Pokeweed Mitogens
PubMed: 37383229
DOI: 10.3389/fimmu.2023.1200146 -
Frontiers in Immunology 2022The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and...
BACKGROUND
The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and T-cell specific response to vaccination.
METHODS
The aim of the study was to evaluate humoral and specific T-cell response, as well as B-cell activation and survival factors, in people with MS (pwMS) under DMTs before (T0) and after two months (T1) from the third dose of vaccine, comparing the obtained findings to healthy donors (HD). All possible combinations of intracellular IFNγ, IL2 and TNFα T-cell production were evaluated, and T-cells were labelled "responding T-cells", those cells that produced at least one of the three cytokines of interest, and "triple positive T-cells", those cells that produced simultaneously all the three cytokines.
RESULTS
The cross-sectional evaluation showed no significant differences in anti-S antibody titers between pwMS and HD at both time-points. In pwMS, lower percentages of responding T-cells at T0 (CD4: p=0.0165; CD8: p=0.0022) and triple positive T-cells at both time-points compared to HD were observed (at T0, CD4: p=0.0007 and CD8: p=0.0703; at T1, CD4: p=0.0422 and CD8: p=0.0535). At T0, pwMS showed higher plasma levels of APRIL, BAFF and CD40L compared to HD (p<0.0001, p<0.0001 and p<0.0001, respectively) and at T1, plasma levels of BAFF were still higher in pwMS compared to HD (p=0.0022).According to DMTs, at both T0 and T1, lower anti-S antibody titers in the depleting/sequestering-out compared to the enriching-in pwMS subgroup were found (p=0.0410 and p=0.0047, respectively) as well as lower percentages of responding CD4+ T-cells (CD4: p=0.0394 and p=0.0004, respectively). Moreover, the depleting/sequestering-out subgroup showed higher percentages of IFNγ-IL2-TNFα+ T-cells at both time-points, compared to the enriching-in subgroup in which a more heterogeneous cytokine profile was observed (at T0 CD4: p=0.0187; at T0 and T1 CD8: p =0.0007 and p =0.0077, respectively).
CONCLUSION
In pwMS, humoral and T-cell response to vaccination seems to be influenced by the different DMTs. pwMS under depleting/sequestering-out treatment can mount cellular responses even in the presence of a low positive humoral response, although the cellular response seems qualitatively inferior compared to HD. An understanding of T-cell quality dynamic is needed to determine the best vaccination strategy and in general the capability of immune response in pwMS under different DMT.
Topics: Humans; Multiple Sclerosis; Tumor Necrosis Factor-alpha; COVID-19 Vaccines; Cross-Sectional Studies; Interleukin-2; COVID-19; Pokeweed Mitogens; Antibodies; Cytokines; RNA, Messenger
PubMed: 36532061
DOI: 10.3389/fimmu.2022.1050183 -
Journal of Applied Physiology... Dec 2019Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is...
Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [, , , and after intensive care unit (ICU) admission] determined in patients with sepsis ( = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, = 10). In a second cohort, expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV ( = 0.01), OKT3 ( = 0.02), and PWM ( = 0.02 on ), whereas unstimulated IFNγ secretion did not differ. expression was also significantly decreased in patients with sepsis at all time points ( = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis. We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.
Topics: Adult; Aged; Cytomegalovirus; Female; Humans; Interferon-gamma; Leukocytes, Mononuclear; Male; Middle Aged; Muromonab-CD3; Pokeweed Mitogens; Sepsis
PubMed: 31545153
DOI: 10.1152/japplphysiol.00438.2019 -
Biology of Sex Differences Apr 2019Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate...
BACKGROUND
Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management.
METHODS
Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1-2 months prior to departure, subsequently every month during their expedition, and 3-4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles.
RESULTS
Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement.
CONCLUSIONS
The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but-with the exception of cortisol and blood cell counts-without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.
Topics: Adult; Antarctic Regions; Antigens, Fungal; Catecholamines; Cytokines; Endocannabinoids; Extreme Environments; Female; Hematologic Tests; Humans; Hydrocortisone; Male; Middle Aged; Pokeweed Mitogens; Sex Characteristics; Stress, Psychological; Young Adult
PubMed: 30992051
DOI: 10.1186/s13293-019-0231-0 -
Scientific Reports Sep 2022Secondary infections have been shown to complicate the clinical course and worsen the outcome of critically ill patients. Severe Coronavirus Disease 2019 (COVID-19) may...
Secondary infections have been shown to complicate the clinical course and worsen the outcome of critically ill patients. Severe Coronavirus Disease 2019 (COVID-19) may be accompanied by a pronounced cytokine release, and immune competence of these patients towards most pathogenic antigens remains uncompromised early in the disease. Patients with bacterial sepsis also exhibit excessive cytokine release with systemic hyper-inflammation, however, typically followed by an anti-inflammatory phase, causing immune paralysis. In a second hit immune response model, leukocyte activation capacity of severely ill patients with pneumonia caused by SARS-CoV-2 or by bacteria were compared upon ICU admission and at days 4 and 7 of the ICU stay. Blood cell count and release of the pro-inflammatory cytokines IL-2, IFNγ and TNF were assessed after whole-blood incubation with the potent immune stimulus pokeweed mitogen (PWM). For comparison, patients with bacterial sepsis not originating from pneumonia, and healthy volunteers were included. Lymphopenia and granulocytosis were less pronounced in COVID-19 patients compared to bacterial sepsis patients. After PWM stimulation, COVID-19 patients showed a reduced release of IFNγ, while IL-2 levels were found similar and TNF levels were increased compared to healthy controls. Interestingly, concentrations of all three cytokines were significantly higher in samples from COVID-19 patients compared to samples from patients with bacterial infection. This fundamental difference in immune competence during a second hit between COVID-19 and sepsis patients may have implications for the selection of immune suppressive or enhancing therapies in personalized medicine.
Topics: COVID-19; Cytokines; Humans; Immunity; Interleukin-2; Pneumonia, Bacterial; Pokeweed Mitogens; SARS-CoV-2; Sepsis
PubMed: 36109525
DOI: 10.1038/s41598-022-17368-9 -
Journal of Clinical Pathology Sep 1993To determine whether the routine measurement of lymphocyte transformation responses to mitogenic stimuli provide any information additional to that available from...
AIMS
To determine whether the routine measurement of lymphocyte transformation responses to mitogenic stimuli provide any information additional to that available from routine T cell CD4 and CD8 analysis in patients with HIV infection.
METHODS
The case records of 197 immunologically investigated HIV seropositive patients were reviewed. The influence of disease stage on T lymphocyte subsets and lymphocyte transformation responses (LyTR) to phytohaemagglutinin (PHA) and Pokeweed mitogen was assessed.
RESULTS
The median CD3 and CD4 counts and LyTR to PHA and Pokeweed mitogen were highest in patients with persistent generalised lymphadenopathy (PGL) and decreased progressively in the order: asymptomatic patients, those with ARC, those with AIDS. LyTR to PHA was preserved in over 70% of all patients, but the response to Pokeweed mitogen was depressed in 8% of patients with PGL, 34% of asymptomatic patients, 68% of those with ARC and 78% of those with AIDS. Subnormal values of both CD4 + T cells and LyTR to Pokeweed mitogen were more common in patients with ARC and AIDS (68%) than in those who were asymptomatic or had PGL (20%).
CONCLUSIONS
CD4 T cell analysis and LyTR to Pokeweed mitogen, but not to PHA, both correlate with disease states in patients with HIV infection.
Topics: AIDS-Related Complex; Acquired Immunodeficiency Syndrome; CD4-CD8 Ratio; CD4-Positive T-Lymphocytes; Female; HIV Infections; Humans; Leukocyte Count; Lymphocyte Activation; Male; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocyte Subsets
PubMed: 7901239
DOI: 10.1136/jcp.46.9.846 -
Scanning 2012B-lymphocyte activation plays an important role in humoral immune system, and its process has been studied well in vivo and in vitro. However, the ultrastructure and...
B-lymphocyte activation plays an important role in humoral immune system, and its process has been studied well in vivo and in vitro. However, the ultrastructure and adhesion property changes remain unclear. In this study, changes in the morphology and mechanical properties of human peripheral blood B lymphocytes were first studied by atomic force microscopy (AFM). B lymphocytes were treated with the mitogen, pokeweed mitogen (PWM), and Staphylococcus aureus Cowan strain I (SAC) for 24 hr. After B lymphocyte is stimulated by the mitogen, the cell height, diameter, and volume are changed in different degree. The ultrastructure of the B lymphocytes membrane obviously displayed proteins gathering, corresponding with larger changes of average roughness and mean height of particles on cell membrane. Meanwhile, we detected the adhesion force of B lymphocytes after being stimulated by PWM and SAC. We found that the treated cells had a higher adhesion force of 304.16 ± 60.30 pN (PWM) and 249.63 ± 58.03 pN (SAC) than that of control group (104.28 ± 21.77 pN). Therefore, our results could provide new information to further understand the B-lymphocyte activation process and their structure-function analyses.
Topics: B-Lymphocytes; Cell Adhesion; Humans; Lymphocyte Activation; Microscopy, Atomic Force; Pokeweed Mitogens; Staphylococcus aureus
PubMed: 21796643
DOI: 10.1002/sca.20258 -
British Medical Journal (Clinical... Aug 1987To identify factors that may predict the development of the acquired immune deficiency syndrome (AIDS) or AIDS related symptoms various immunological measurements were...
To identify factors that may predict the development of the acquired immune deficiency syndrome (AIDS) or AIDS related symptoms various immunological measurements were studied in a group of homosexual men attending screening clinics for AIDS in Copenhagen. Fifty seven men whose ratio of T helper lymphocytes to T suppressor lymphocytes (CD4:CD8 ratio) was less than 1.0 before the study began were included. Forty two were positive for antibody to the human immunodeficiency virus (HIV), of whom 38 were reinvestigated after a median observation period of 10 months. Among the seropositive men the transformation responses to pokeweed mitogen and cytomegalovirus and the absolute count of CD4 positive lymphocytes were the most common abnormal values. In particular, a low relative response to pokeweed mitogen on initial investigation correlated with a worsened clinical condition on reinvestigation. The risk of a worsened clinical condition was 55 times higher in seropositive men whose responses to pokeweed mitogen were low than in other seropositive men. The corresponding relative risks for low transformation responses to cytomegalovirus and for a decreased absolute count of CD4 positive lymphocytes were 18 and six. The relative response to pokeweed mitogen is therefore a very sensitive short term predictive marker of the clinical condition of seropositive patients who have a CD4:CD8 ratio of less than 1.0.
Topics: AIDS-Related Complex; Acquired Immunodeficiency Syndrome; Adult; Antibodies, Viral; Antigens, Surface; Antigens, Viral; Cytomegalovirus; HIV; Homosexuality; Humans; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Pokeweed Mitogens; Prognosis
PubMed: 2820544
DOI: 10.1136/bmj.295.6593.293