scholarly journals B Cells in Health and Disease – Leveraging Flow Cytometry to Evaluate Disease Phenotype and the Impact of Treatment with Immunomodulatory Therapeutics

10.5772/38288 ◽  
2012 ◽  
Author(s):  
Cherie L. ◽  
John Ferbas ◽  
Barbara A.
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Disease Phenotype ◽  
Health And Disease ◽  
The Impact

Tumor Cells Resistant to Alemtuzumab Complement Mediated Cytotoxicity in Patients with High Risk Previously Untreated Early Stage CLL: A Possible Mechanism of Treatment Failure.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2973-2973
Author(s):  
Clive S. Zent ◽  
Nancy D. Bone ◽  
Susan M. Geyer ◽  
Neil E. Kay
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
High Risk ◽  
B Cell ◽  
Cell Clone ◽  
Early Stage ◽  
Cell Membrane Permeability ◽  
Viable Cells ◽  
The Impact

Abstract The monoclonal antibodies (MoAb) alemtuzumab and rituximab have proven efficacy in the treatment of CLL. In addition, alemtuzumab is effective in patients with defective p53 function responding poorly to purine analogue therapy. The action of both MoAb is not completely understood. Proposed mechanisms include complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and direct induction of apoptosis of CLL B cells. We have done correlative studies on CLL B cells from patients enrolled in a trial of alemtuzumab and rituximab in “high risk” early stage previously untreated CLL to determine: 1. Role of apoptosis induction and CDC in each MoAb and 2. If the addition of rituximab to alemtuzumab increases their in vitro cytotoxicity. Patients and Methods: Patients with early stage, previously untreated, high risk CLL are treated with subcutaneous alemtuzumab (dose escalation over 3 days then 30 mg Mon-Wed-Fri for 4 weeks) and rituximab (375 mg/m2/dose weekly from day 8 x 4 doses). High risk disease was defined as one or more of the following features of the CLL B cell clone: (1) 17p13−; (2) 11q22−; (3) unmutated IgVH (< 2%) and either CD38+ or ZAP-70+. Blood B lymphocytes collected prior to the start of therapy were tested for response to MoAb in vitro. Cells were cultured at 2 x 106/ml in AIM-V medium using standard conditions. Alemtuzumab and rituximab were used at 20 μg/ml and complement as 10% of 40 CH50 units/ml human serum. The impact of the MoAb was measured by counting viable cells (trypan blue negative) and measuring early apoptosis (annexin V) and cell death (cell membrane permeability to propidium iodide) using flow cytometry at 1 hour, and then daily for 3 days. Results: Treatment caused rapid resolution of lymphocytosis in all 7 patients and 3 patients were negative for circulating CLL cells using a highly sensitive 3 color flow cytometry (CD5+/CD19+/CD79b-) after therapy. All patients had a clinical response (2 CR, 5 PR). Alemtuzumab and complement were rapidly cytotoxic to most CLL cells. Mean cell viability was 39% (sd: 8%) after 1 hour of incubation. Cytotoxicity was similar in all samples irrespective of FISH defects, IgVH mutation status, and in vitro resistance to F-ara-A (n = 3). Alemtuzumab was inactive in the absence of complement for all samples. Rituximab alone and together with complement did not induce cytotoxicity or apoptosis. However, the addition of rituximab to alemtuzumab and complement did increase CDC where the number of viable cells was significantly lower at 1, 24, 48, and 72 hours incubation (p = 0.075, 0.047, 0.031, 0.027, respectively, for pairwise comparisons). CLL cells surviving alemtuzumab CDC subsequently had a lower level of apoptosis than control cells, implying a selection for resistant cells. Alemtuzumab CDC on this residual population was not increased at higher concentrations of alemtuzumab or complement. This mechanism of CDC resistance is currently under investigation. Conclusion: These data suggest that alemtuzumab CDC is an important mechanism of action in patients with CLL. However, alemtuzumab CDC kills only about 61% of CLL cells in vitro, and the surviving cells are more resistant to spontaneous apoptosis. This suggests that cells that survive alemtuzimab CDC contribute to disease progression or relapse. We intend to elucidate the mechanism of this resistance using our in vitro model with the hope that treatment strategies can be deployed to remove this residual CLL B cell clone.

Patients With DLBCL Commonly Have B-Cell Lymphopenia and Circulating Clonal B-Cell Populations With a Phenotype Concordant With The Underlying Tumour

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3013-3013
Author(s):  
Ruth M de Tute ◽  
Sharon Barrans ◽  
Andy C. Rawstron ◽  
Peter W.M. Johnson ◽  
Andrew J Davies ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Peripheral Blood ◽  
Expression Profiling ◽  
Common Finding ◽  
Cell Populations ◽  
The Impact

Abstract Clonal B-cell populations with either a CLL or a non-CLL phenotype are a common finding in normal individuals but uncertainty remains about how this relates to the development of clinically significant disease. The aim of this study was to investigate the frequency of peripheral blood clonal B-cell populations and B-cell subset abnormalities in newly presenting DLBCL patients and to determine whether the incidence of these abnormalities differed between the GCB and ABC subtypes, which are regarded as having distinct pathogenesis. The study was carried out using peripheral blood samples collected from patients entered in the UK-REMoDL-B trial. This trial is testing the hypothesis that the ABC subtype of DLBCL responds preferentially to R-CHOP- Bortezomib. Gene expression profiling is performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay prior to randomisation classified as GCB, ABC or unclassified (UN). The availability of GEP data allows meaningful comparison with the phenotype of clonal populations detected by flow cytometry. Peripheral blood taken prior to first treatment was analysed using multi-colour flow cytometry. Following red cell lysis with ammonium chloride, samples were incubated with a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. A minimum of 500,000 events were acquired on a FacsCanto II flow cytometer (Becton Dickinson). B-cells were enumerated and any monoclonal populations identified were classified as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. 358 samples were eligible for inclusion from patients with an average age of 62.2years (range 22.9-86.1). Abnormalities were detected in 52% of cases (B-lymphopenia ((<0.06 x 109/l) 33%, B-lymphocytosis (>1 x 109/l) 2.8%, CLL clone 3.6%, GC clone 9.8%, non-GC clone 9.8%, clonal population NOS 2.2%). Gene expression profiling results were available for 278 individuals; 51% GCB, 32% ABC and 17% unclassified. The relationship between peripheral blood B-cell findings and the GEP determined phenotype of the tumour is shown in the table:TableB-lymphopeniaCLL CloneMonoclonal GC typeMonoclonalNon-GC typeMonoclonal NOSNormalB-cellGCB n=14241/142 (29%)5/142 (3.5%)21/142 (15%)8/142 (5.6%)2/142 (1%)72/142 (51%)ABC n=8927/89 (30%)2/89 (2%)2/89 (2%)12/89 (13.5%)2/89 (2%)49/89 (55%)Unclassified n=4726/47 (55%)0/50 (0%)2/47 (4%)6/47 (12%)6/47 (5%)14/47 (30%) In patients where clonal populations were detected in the peripheral blood there was striking concordance between the phenotype of the clone and the GEP of the underlying tumour. Presence of a GC-population by flow was highly predictive of GCB GEP (84% GC–type populations detected were in GCB cases). The number of discordant cases and the number of CLL clones detected approximate to the numbers that would be expected in a normal population of a similar age. It is, therefore, likely that in most cases circulating tumour cells or a closely related precursor clone are being detected. The similarity between the results of the ABC and unclassified GEP groups suggest that these are biologically related. An unexpected finding in this study was the high incidence of B-lymphopenia at a level that might be expected to be associated with increased risk of infection. This may reflect suppression of normal B-cells by the neoplastic clone or be a marker of underlying immune dysfunction that may predispose to the development of the tumour. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and this study suggests that this may also be a factor in the wider patient population. These results may have implications for prognostic assessment and may offer opportunities for early diagnosis and possibly response assessment in some patients. The impact on outcome will be assessed in the course of the trial. Disclosures: Jack: Roche /Genentech: Research Funding.

The Tumor Microenvironment Measured by Flow Cytometry Predicts Overall Survival (OS) and Transformation Risk (TR) in Follicular Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2406-2406 ◽  
Author(s):  
Pedro Farinha ◽  
John Han ◽  
Abdulwahab Al-Tourah ◽  
Joseph M. Connors ◽  
Diponkar Banerjee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
Light Chain ◽  
Clinical Event ◽  
Light Chains ◽  
Biological Variables ◽  
The Impact

Abstract Background: FL is an indolent but heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma is a dominant clinical event that is frequently followed by shorter survival. There is no consistent biological prognostic marker for TR. Recent studies have highlighted the role of the microenvironment in helping to determine the prognosis of FL. However, its impact on TR is largely unknown. In this study we used diagnostic flow cytometry (FC) analysis to assess the role of non-malignant cells in determining OS and TR in FL. Methods: We identified 567 patients with FL diagnosed at the BCCA over a 5-year period between 1997 and 2001. Of these, 270 cases had diagnostic FC and histological review, of which 137 were nodal and had complete clinical data in our electronic database. FC results were re-analyzed. The antibodies studied included anti-CD3, CD4, CD5, CD8, CD10, CD14, CD19, CD20, CD23, CD45, FMC-7 and IG kappa and lambda light chains. To ensure that biopsies were representative, the sum of the % gated events for CD3 and CD20 had to equal 100% +/− 20%. Light chain restriction was present in all cases and established clonality. The estimate of neoplastic B cells was determined by examining CD19 and CD20 frequency and the ratio of clonal light chain vs non-clonal light chain. Non-neoplastic B cells were estimated using CD19/20 and the amount of non-clonal light chain × 0.5 (λ clonal) or × 2 (κ clonal). Clinical characteristics, different subsets of T cells and the cell content of reactive, non-neoplastic B cells were evaluated using SPSS® software. Results: The median age of the 137 patients was 57 years, 51.8% were male and 38.6% had a high IPI (4/5). There were 97 grade 1, 27 grade 2 and 13 grade 3a FL. The median ratio of CD4/CD8 was 4.4. Patients were given a variety of treatments, including observation if asymptomatic, precluding an analysis of progression-free survival. The median follow-up of the living patients was 5.8 years and the estimated 5-year OS and TR were 70% and 20%, respectively. The IPI was predictive of OS (p<0.0001). Two biological variables showed a significant impact on survival. Firstly, cases in which CD8+ cells represented more than 25% of the total (CD3+) T cells had shorter OS (p = 0.028) and increased TR (p = 0.013). The CD8 ratio (p = 0.026) affected OS independently of IPI (p = 0.046). Secondly, cases with a low content of reactive, non-neoplastic B cells, defined by the ratio between IG light chains >1/15 had shorter OS (p = 0.003) and increased TR (p=0.002). The impact of a reduction in normal reactive B cells (p = 0.014) on transformation risk was independent of the IPI (p = 0.05). Conclusion: Two features of the FL microenvironment studied by diagnostic FC demonstrated an impact on prognosis. The proportion of CD8+ T cells relative to the total T cells and the number of residual, non-neoplastic B cells were both predictors of OS. Importantly, both predict, independently of the IPI, the risk of transformation. These biomarkers are easily measured and may be used to better stratify patients, choose initial treatment options and predict transformation risk in patients with FL. Microenvironment & Transformation Risk in Follicular Lymphoma Microenvironment & Transformation Risk in Follicular Lymphoma

Drug Induced Changes in Cell Organelles

1968 ◽  
Vol 26 ◽  
pp. 110-111
Author(s):  
Sarah A. Luse
Keyword(s):  
Clinical Medicine ◽  
Cell Organelles ◽  
Riboflavin Deficiency ◽  
Drug Induced ◽  
New Era ◽  
Health And Disease ◽  
In The Beginning ◽  
Cellular Pathology ◽  
Induced Changes ◽  
The Impact

In the mid-nineteenth century Virchow revolutionized pathology by introduction of the concept of “cellular pathology”. Today, a century later, this term has increasing significance in health and disease. We now are in the beginning of a new era in pathology, one which might well be termed “organelle pathology” or “subcellular pathology”. The impact of lysosomal diseases on clinical medicine exemplifies this role of pathology of organelles in elucidation of disease today.Another aspect of cell organelles of prime importance is their pathologic alteration by drugs, toxins, hormones and malnutrition. The sensitivity of cell organelles to minute alterations in their environment offers an accurate evaluation of the site of action of drugs in the study of both function and toxicity. Examples of mitochondrial lesions include the effect of DDD on the adrenal cortex, riboflavin deficiency on liver cells, elevated blood ammonia on the neuron and some 8-aminoquinolines on myocardium.

The Laryngeal Epithelium in Reflux

2011 ◽  
Vol 21 (3) ◽  
pp. 112-117 ◽  
Author(s):  
Elizabeth Erickson-Levendoski ◽  
Mahalakshmi Sivasankar
Keyword(s):  
Laryngopharyngeal Reflux ◽  
Critical Role ◽  
Structure And Function ◽  
Laryngeal Disease ◽  
Health And Disease ◽  
And Function ◽  
The Impact

The epithelium plays a critical role in the maintenance of laryngeal health. This is evident in that laryngeal disease may result when the integrity of the epithelium is compromised by insults such as laryngopharyngeal reflux. In this article, we will review the structure and function of the laryngeal epithelium and summarize the impact of laryngopharyngeal reflux on the epithelium. Research investigating the ramifications of reflux on the epithelium has improved our understanding of laryngeal disease associated with laryngopharyngeal reflux. It further highlights the need for continued research on the laryngeal epithelium in health and disease.

B-cell and T-cell values in peripheral blood in Polish mixed-breed rabbits with addition of blood of meet breeds

2014 ◽  
Vol 17 (3) ◽  
pp. 421-426 ◽  
Author(s):  
B. Tokarz-Deptuła ◽  
P. Niedźwiedzka-Rystwej ◽  
B. Hukowska-Szematowicz ◽  
M. Adamiak ◽  
A. Trzeciak-Ryczek ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Laboratory Animals ◽  
Immunological Parameters ◽  
Four Seasons ◽  
Mixed Breed ◽  
The Impact

Abstract In Poland, rabbit is a highly valued animal, due to dietetic and flavour values of its meat, but above all, rabbits tend to be commonly used laboratory animals. The aim of the study was developing standards for counts of B-cells with CD19+ receptor, T-cells with CD5+ receptor, and their subpopulations, namely T-cells with CD4+, CD8+ and CD25+ receptor in the peripheral blood of mixed-breed Polish rabbits with addition of blood of meet breeds, including the assessment of the impact of four seasons of the year and animal sex on the values of the immunological parameters determined. The results showed that the counts of B- and T-cells and their subpopulations in peripheral blood remain within the following ranges: for CD19+ B-cells: 1.05 - 3.05%, for CD5+ T-cells: 34.00 - 43.07%, CD4+ T-cells: 23.52 - 33.23%, CD8+ T-cells: 12.55 - 17.30%, whereas for CD25+ T-cells: 0.72 - 2.81%. As it comes to the season of the year, it was observed that it principally affects the values of CD25+ T-cells, while in the case of rabbit sex, more changes were found in females.

scholarly journals BOARD INVITED REVIEW: The pig microbiota and the potential for harnessing the power of the microbiome to improve growth and health1

2019 ◽  
Vol 97 (9) ◽  
pp. 3741-3757 ◽  
Author(s):  
Nirosh D Aluthge ◽  
Dana M Van Sambeek ◽  
Erin E Carney-Hinkle ◽  
Yanshuo S Li ◽  
Samodha C Fernando ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Gut Microbiota ◽  
Therapeutic Potential ◽  
Animal Health ◽  
Microbial Colonization ◽  
Critical Importance ◽  
Specific Host ◽  
Microbiome Research ◽  
Health And Disease ◽  
The Impact

Abstract A variety of microorganisms inhabit the gastrointestinal tract of animals including bacteria, archaea, fungi, protozoa, and viruses. Pioneers in gut microbiology have stressed the critical importance of diet:microbe interactions and how these interactions may contribute to health status. As scientists have overcome the limitations of culture-based microbiology, the importance of these interactions has become more clear even to the extent that the gut microbiota has emerged as an important immunologic and metabolic organ. Recent advances in metagenomics and metabolomics have helped scientists to demonstrate that interactions among the diet, the gut microbiota, and the host to have profound effects on animal health and disease. However, although scientists have now accumulated a great deal of data with respect to what organisms comprise the gastrointestinal landscape, there is a need to look more closely at causative effects of the microbiome. The objective of this review is intended to provide: 1) a review of what is currently known with respect to the dynamics of microbial colonization of the porcine gastrointestinal tract; 2) a review of the impact of nutrient:microbe effects on growth and health; 3) examples of the therapeutic potential of prebiotics, probiotics, and synbiotics; and 4) a discussion about what the future holds with respect to microbiome research opportunities and challenges. Taken together, by considering what is currently known in the four aforementioned areas, our overarching goal is to set the stage for narrowing the path towards discovering how the porcine gut microbiota (individually and collectively) may affect specific host phenotypes.

scholarly journals THU0231 IL-2 DRIVES THE CONVERSION OF T FOLLICULAR HELPER TO T FOLLICULAR REGULATORY CELLS THROUGH EPIGENETIC MODIFICATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 343.2-343
Author(s):  
H. Hao ◽  
S. Nakayamada ◽  
Y. Kaoru ◽  
N. Ohkubo ◽  
S. Iwata ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
B Cells ◽  
Histone Modifications ◽  
Lupus Erythematosus ◽  
Regulatory Cells ◽  
Systemic Lupus ◽  
Research Support ◽  
Tfh Cells ◽  
Follicular Helper

Background:Systemic lupus erythematosus (SLE) is a complex polygenic autoimmune disease characterized by immune-system aberrations. Among several types of immune cells, T follicular helper (Tfh) cells promote autoantibody production, whereas T follicular regulatory (Tfr) cells suppress Tfh-mediated antibody responses.(1)Objectives:To identify the characteristics of Tfr cells and to elucidate the mechanisms of conversion of Tfh cells to Tfr cells, we probed the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors.Methods:Peripheral blood mononuclear cells from SLE patients (n=44) and healthy donors (HD; n=26) were analyzed by flow cytometry. Memory Tfh cells were sorted and cultured under stimulation with T cell receptor and various cytokines. Expression of characteristic markers and phosphorylation of STATs (p-STATs) were analyzed by flow cytometry and quantitation PCR. Histone modifications were evaluated by chromatin immunoprecipitation.Results:The proportion of CXCR5+FoxP3+Tfr cells in CD4+T cells tended to increase (2.1% vs 1.7%, p=0.17); however, that of CD4+CD45RA-FoxP3hiactivated Tfr cells in Tfr cells was decreased (4.8% vs 7.1%, p<0.05), while CD4+CD45RA-FoxP3lownon-suppressive Tfr cells was increased (50.1% vs 38.2%, p<0.01) in SLE compared to HD. The percentage of PD-1hiactivated Tfh cells was significantly higher in SLE compared to HD (15.7% vs 5.9%, p<0.01). Furthermore, active patients had a higher ratio of activated Tfh/Tfr cells compared to inactive patients. In vitro study showed that IL-2, but not other cytokines such as TGF-β1, IL-12, IL-27, and IL-35, induced the conversion of memory Tfh cells to functional Tfr cells characterized by CXCR5+Bcl6+Foxp3hipSTAT3+pSTAT5+cells. The loci ofFOXP3at STAT binding sites were marked by bivalent histone modifications. After IL-2 stimulation, STAT5 directly bound on FOXP3 gene loci accompanied by suppressing H3K27me3. Finally, we found that serum level of IL-2 was decreased in SLE and that stimulation with IL-2 suppressed the generation of CD38+CD27+B cells by ex vivo coculture assay using Tfh cells and B cells isolated from human blood.Conclusion:Our findings indicated that the regulatory function of Tfr cells is impaired due to the low ability of IL-2 production and that IL-2 restores the function of Tfr cells through conversion of Tfh cells to Tfr cells in SLE. Thus, the reinstatement of the balance between Tfh and Tfr cells will provide important therapeutic approaches for SLE.References:[1]Deng J, Wei Y, Fonseca VR, et al. T follicular helper cells and T follicular regulatory cells in rheumatic diseases. Nat Rev Rheumatol. 2019; 15 (8): 475-90.Disclosure of Interests: :He Hao: None declared, Shingo Nakayamada Grant/research support from: Mitsubishi-Tanabe, Takeda, Novartis and MSD, Speakers bureau: Bristol-Myers, Sanofi, Abbvie, Eisai, Eli Lilly, Chugai, Asahi-kasei and Pfizer, Yamagata Kaoru: None declared, Naoaki Ohkubo: None declared, Shigeru Iwata: None declared, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin

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