Lymphocyte Surface Antigens Research Articles

Novel diagnostic method for B cell vitreoretinal lymphoma by identification of regulatory T cells and PD-1+ cytotoxic T lymphocytes in the vitreous via flow cytometry

AimsTo investigate the significance of regulatory T cells (Tregs) and programmed cell death 1 (PD-1)+ cytotoxic T lymphocytes (CTLs) in the vitreous of patients with vitreoretinal lymphoma (VRL) and uveitis.MethodsThis...

CD38 Deficiency Protects Mice from High Fat Diet-Induced Nonalcoholic Fatty Liver Disease through Activating NAD+/Sirtuins Signaling Pathways-Mediated Inhibition of Lipid Accumulation and Oxidative Stress in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. CD38 was initially identified as a lymphocyte surface antigen and then has been found to exist in a variety of cell types. Our previous studies showed that CD38-/- mice were resistant to high-fat diet (HFD)-induced obesity. However, the role and mechanism of CD38 in HFD-induced NAFLD is still unclear. Here, we reported that CD38-/- mice significantly alleviated HFD-induced hepatic steatosis. HFD or oleic acid (OA) remarkably increased the mRNA and protein expressions of CD38 in mouse hepatic tissues and primary hepatocytes or hepatic cell lines in vitro and in vivo, suggesting that CD38 might play a role in HFD-induced hepatic steatosis. We observed that CD38 deficiency markedly decreased HFD- or OA-induced the lipid accumulation and oxidative stress in CD38-/- livers or primary hepatocytes, respectively. In contrast, overexpression of CD38 in Hep1-6 cells aggravated OA-induced lipid accumulation and oxidative stress. Furthermore, CD38 deficiency markedly inhibited HFD- or OA-induced the expressions of NOX4, and increased the expression of PPARα, CPT1, ACOX1 and SOD2 in liver tissue and hepatocytes from CD38-/- mice, indicating that CD38 deficiency-mediated the enhancement of fatty acid oxidation and the inhibition of oxidative stress contributed to protecting NAFLD. More importantly, Ex527 (Sirt1 inhibitor) and 3-TYP (Sirt3 inhibitor) significantly enhanced OA-induced lipid accumulation and oxidative stress in CD38-/- primary hepatocytes, suggesting that the anti-lipid accumulation of CD38 deficiency might be dependent on NAD/Sirtuins-mediated enhancement of FAA β-oxidation and suppression of oxidative stress in hepatocytes. In conclusion, we demonstrated that CD38 deficiency protected mice from HFD-induced NAFLD by reducing lipid accumulation and suppressing oxidative stress via activating NAD/Sirtuins signaling pathways.

Plasmacytoid dendritic cells (pDCs) and IFN-α production in aged squirrel monkeys (Saimiri boliviensis boliviensis) stimulated with Class C CpG Oligodeoxynucleotides

Abstract Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs and immunomodulatory properties signal through an endosomal membrane-based type of pattern recognition receptor (PRR), the Toll-like receptor 9 (TLR9), that has been identified in dendritic cells (DCs), B cells, and other human immune cell types. CpG-ODNs influence several signaling pathways, leading to cytokine production in many mammalian species that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions. The squirrel monkey (SQM), a well-established New World non-human primate (NHP) model is known to be used for studying various diseases such as Alzheimer’s disease (AD), malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). In the present study, we generated the first report of immunoregulatory activity of class C CpG-ODN in aged squirrel monkey. CpG-ODN exhibits very potent effects on squirrel monkey immune cells by inducing IFN-γ/IFN-α from peripheral blood mononuclear cells and demonstrates accelerated kinetics of activity. In addition, class C CpG-ODN specifically activates pDCs to undergo maturation and secrete cytokines, including high levels of IFN-α. To best to our knowledge, this is the first documentation describing immune status in squirrel monkeys immunized with C class CpG-ODN by concentrating on lymphocyte surface antigen expression and function. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG-ODN in this NHP model allowing for future therapeutic trials of innate immunity stimulation via TLR9 agonists for diverse indications including AD therapeutics.

Status of cellular immunity in rats under conditions of acute widespread petitonitis in the setting of diabetes mellitus

Acute generalized peritonitis (AGP) occurs in patients with concomitant pathology, in particular at diabetes mellitus. The severity of peritonitis depends on the adequacy of the immune response. Purpose of research was to study the features of cellular arm of immune response in the organism of experimental animals with simulated AGP in the setting of diabetes mellitus (DM). 56 white rats were used in the experiment, which was divided into three groups: main group – 24 animals with simulated AWP in the setting of DM; comparison group – 24 animals with simulated peritonitis. Animals of the main group AGP were modelled by injecting 10 % of filtered fecal suspension into the abdominal cavity of tested rats at a dose of 0,5 ml per 100 g of body weight. Removal of material for histological examination was performed on 1, 3 and 7 days. Cellular immunity was determined by a method based on the interaction of fluorescently labeed monoclonal antibodies with lymphocyte surface antigens. In both animals, all indicators of cellular immunity gradually decreased from day 1 to day 7 of the experiment, but these changes were more significant in animals with DM. With AGP, the cellular immune response imbalance is more significant in animals with DM than in animals with isolated AGP, which is characterized by a marked statistically significant decrease in the level of CD3+ cells, CD4+ cells, CD16+ cells, and a moderate increase in CD8+ cells. The imbalance of cellular immunity deepens depending on the duration of the lesion. The level of CD3+ cells at 1, 3, and 7 day in animals with AGP in the setting of DM was 53,77%, 60,48%, and 62,1 %, respectively, lower than the level in the group with animals with AGP. The prolonged imbalance of cellular immunity indices in experimental animals with simulated AGP in the setting of DM indicates not only the occurrence of secondary immunodeficiency, but also significant depletion of the body’s immune forces compared to animals with AGP.

Dynamics of indicators of cellular immunity in conditions of acute generalized peritonitis in rats

The course of peritonitis depends on the state of the immune system and the adequacy of the immune response. The aim of this investigation was to explore the features of the cellular immune response cell in rats with simulated acute generalized peritonitis (AGP). The study was conducted on 32 rats, divided into two groups: the main group – 24 animals with simulated peritonitis; control– 8 intact animals. In the animals of the main group AGP were modeleted by injecting of 10 % filtered stool suspension into the abdominal cavity of the study rats at a dose of 0.5 ml per 100 g of body weight. Removal of material for histological examination was carried out on days 1, 3 and 7. Indicators of cellular immunity were determined by a method that was based on the interaction of fluorescently labeled monoclonal antibodies with lymphocyte surface antigens. In the main group of animals, all indicators of the cellular immunity gradually decreased from 1 to 7 days of the experiment. The concentration of CD3+ - cells decreased by 1.90 times per day, by 1.97 times by 3 days, and by 2.10 times by 7 days, compared with intact animals. Suppression of the cellular immunity was observed after modeling of AGP, which is combined with a decrease in the number of both CD3+ lymphocytes and the main subpopulations of CD4+, CD8+ and CD16+ cells. The greatest decrease in the cellular level of immunity was observed on day 7 of the experiment.

Effect Of Self control Exercise On Lymphocyte Subsets Of Lung Cancer Patients During Rehabilitation

PURPOSE: Self-Control Exercise (SCE), known also as Guolin Qigong, is a mind-body exercise being used in China for cancer survival for more than 40 years. This study was to examine the effect of SCE on lymphocyte subsets of lung cancer patients and the possible mechanisms. METHODS: 26 lung cancer patients (8 males & 18 females; Age in yr: 60.08±5.41; Cancer survival yr: 1.69±0.72) were recruited from the Shanghai Cancer Club. All the patients were diagnosed pathologically. The patients began to learn SCE for 3 weeks and then performed 6 months SCE at their will. Cancer history was surveyed, physical activity including SCE was recorded during the intervention. The lymphocyte surface antigen CD3/CD4/CD8/CD28/CD(16+56)/CD19/CD4CD25Foxp3 were examined by direct immunofluorescence staining and flow cytometry. Paired-samples t test was performed to compare the change of lymphocyte surface antigen before and after the intervention. RESULTS: It was found that CD3+ increased significantly (50.52±13.09, 56.77±11.56 p &lt 0.05), CD4+ (22.54±6.70, 33.21±7.55) and CD4+/CD8+ (1.05±0.46, 1.80±0.69) increased significantly (p &lt 0.01), CD4+CD25+Foxp3 declined significantly (18.99±8.55, 13.04±5.86, p &lt 0.05), CD16+CD56+ increased significantly (17.58±8.35, 21.48±8.81, p &lt 0.05) after the intervention. CD8+, CD8+CD28+ and CD19+ showed no statistical difference (p &gt 0.05) before and after intervention. CONCLUSIONS: SCE intervention improved the cellular immune function which mediated by T cell and NK cell, and it may be related to the suppression and down-regulation of the T regulatory cells to the cell immunity.

Pharmacodynamic Monitoring of mTOR Inhibitors

Pharmacodynamic monitoring may complement routine pharmacokinetic monitoring of mTOR inhibitors (mTORi) in an attempt to better guide individualized sirolimus (SRL) or everolimus (EVR) treatment after organ transplantation. This review focuses on current knowledge about pharmacodynamic biomarkers for personalized mTORi therapies.Different strategies have already been employed in the evaluation of the pharmacodynamics of SRL and EVR as a proxy for their effects on the immune response after transplantation. These include measuring p70S6K (70 kDa ribosomal protein S6 kinase) activity, p70S6K phosphorylation (P-p70S6K), or P-S6 protein expression. Compared to Western blot and ELISA, phosphoflow cytometry can detect phosphorylated proteins and differentiate activation-induced changes of signaling molecules inside the cell from unstimulated populations of identical cells in the same sample. Alternatively, in patients receiving a combined therapy, other pharmacodynamic approach is to consider biomarkers such as NFAT residual expression for calcineurin inhibitors, or to evaluate non-specific effects of the drugs such as lymphocyte proliferation, interleukin synthesis, specific peripheral blood T regulatory subsets or lymphocyte surface antigens, which have the advantage to reflect the overall immunosuppressive status achieved. Although limited, the available data on mTOR pathway biomarkers seem promising. Before clinical implementation, the analytical methodologies must be standardized and cross-validated, and the selected biomarkers will have to demonstrate their clinical utility for SRL or EVR dose individualization in multicenter clinical trials.

Unusual nuclear form hairy cells in hairy cell leukemia discovered using a lymphocyte-binding anti-CD antibody microarray.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder constituting about 2% from all leukemia cases and characterized by typical “hairy” morphology of tumor lymphocytes. We describe an HCL case with atypical nuclear shapes (lymphocytes with clover-leaf-like, horse-shoe-like, ring-shaped nuclei and binuclear cells were present). Morphology and immunophenotype of circulating leukemic cells were studied using a cell-binding microarray - a transparent plastic slide with immobilized monoclonal antibodies against surface antigens of lymphocytes. The cell-binding microarray with immobilized anti-CD11c, anti-CD103 and anti-CD123 permits to study a lymphocyte population enriched with hairy cells. Hairy cells with atypical nuclei constituted 3% of all lymphocytes and 15% of all hairy cells. This unusual hairy cell morphology is the first described in Russia and was found in one out of 85 HCL cases in our practice.

Two-way detection of image features and immunolabeling of lymphoma cells with one-step microarray analysis.

Detecting the number of pathological lymphoma cells and lymphocyte subtypes in blood is helpful for clinical diagnosis and typing of lymphoma. In the current study, cell type is identified by cell morphological features and immunolabeled lymphocyte subtypes. Red blood cells and leukocytes were separated using a microfluidic cell chip based on physical blood cell parameters, and leukocytes were identified using five characteristic parameters: energy variance, entropy variance, moment of inertia variance, color mean, and cell area individually. The number of red blood cells that could come into contact with the leukocyte membrane was ≤2 based on the microfluidic injection flow rate of microfluidic chips. Anti-CD3 and anti-CD19 antibodies were used for immunofluorescence staining of T-lymphocyte and B-lymphocyte surface antigens, respectively. The results suggested that the microfluidic assay could detect lymphocyte surface antigen markers and intact leukocytes. Therefore, we report a one-step microfluidic chip for classifying hematological lymphoma cells based on the physical parameters of cells, which can simultaneously measure the overall morphology of blood cells and immunolabeling of lymphocyte surface antigens in one step, solving the current problem of detecting subtypes of hematological lymphoma cells based on multiple methods and multi-step detection.

Serum biomarkers of glucocorticoid response and safety in anti-neutrophil cytoplasmic antibody-associated vasculitis and juvenile dermatomyositis

Glucocorticoids are standard of care for many chronic inflammatory conditions, including juvenile dermatomyositis (JDM) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We sought to define pharmacodynamic biomarkers of therapeutic efficacy and safety concerns of glucocorticoid treatment for these two disorders. Previous proteomic profiling of patients with Duchenne muscular dystrophy (DMD) and inflammatory bowel disease (IBD) treated with glucocorticoids identified candidate biomarkers for efficacy and safety concerns of glucocorticoids. Serial serum samples from patients with AAV (n = 30) and JDM (n = 12) were obtained during active disease, and after treatment with glucocorticoids. For AAV, 8 of 11 biomarkers of the anti-inflammatory response to glucocorticoids were validated (P-value ≤0.05; CD23, macrophage-derived cytokine, interleukin-22 binding protein, matrix metalloproteinase-12, T lymphocyte surface antigen Ly9, fibrinogen gamma chain, angiopoietin-2 [all decreased], and protein C [increased]), as were 5 of 7 safety biomarkers (P-value ≤0.05; afamin, matrix metalloproteinase-3, insulin growth factor binding protein-5, angiotensinogen, leptin [all increased]). For JDM, 10 of 11 efficacy biomarkers were validated (P-value ≤0.05; all proteins except fibrinogen gamma chain) and 6 of 7 safety biomarkers (P-value ≤0.05; AAV proteins plus growth hormone binding protein). The identified efficacy biomarkers may be useful as objective outcome measures for early phase proof-of-concept studies when assessing novel anti-inflammatory drugs in JDM and AAV, and likely in other inflammatory disorders. Similarly, safety biomarkers may also be helpful assessing toxicity of alternatives to glucocorticoids.

Abstract B18: Noninvasive imaging of antitumor immune responses

Abstract By using the body's own immune response, immunotherapy may prove to be a turning point in the treatment of cancer. Predictors of a patient's response to immunotherapy remain to be defined, as actual responses are heterogeneous and difficult to study in real-time. Is it possible to predict the outcome of therapy for an individual patient? What is the behavior and what are the dynamics of immune cells in the tumor microenvironment? We need new methods to noninvasively explore the microenvironment of tumors and enumerate relevant cell types. Imaging the extent of infiltration of immune cells into tumors and identifying the relevant lymphocyte subsets is therefore essential. We developed single domain antibodies (VHHs, ~15 kDa) against a variety of lymphocyte surface antigens to image antitumor immune responses. VHHs were engineered to contain a sortase recognition tag, X-LPETGG, at their C-terminus to enable site-specific modification with tags of choice. We synthesized a substrate that contains both a click handle and a metal chelator, where the click handle can be used to attach a polymer, and the chelator is used to chelate a radiometal onto the VHH of interest. We thus produced VHHs equipped with a polyethyleneglycol (PEG) molecule to increase circulatory half-life and a chelator. C57/BL6 mice were injected with 89Zr-PEGylated VHHs, and images were acquired 24 h post injection. Images of the PEGylated-VHHs showed specific staining with low background in kidney and intestine. When we used 89Zr-labeled PEGylated-VHHs specific for CD8 or Class II MHC, secondary lymphoid structures, including the mesenteric lymph node, were clearly visible. Knock-out mice were used to establish specificity of PET imaging. Mice bearing panc02 tumors were imaged at 24 h p.i. and showed CD8 T cells infiltrating the tumor. We monitored the CD8-T cell response to immunotherapy in the B16 melanoma model by using the antiCD8 VHH. For animals that showed a response that led to regression of the tumor, the distribution of the signal was homogeneous throughout the tumor. It is thus possible to monitor antitumor immune responses. Noninvasive monitoring could therefore change how therapy is applied. Citation Format: Mohammad Rashidian, Michael Dougan, Jessica Ingram, Hidde Ploegh. Noninvasive imaging of antitumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B18.

A Canine Model of Chronic Graft-versus-Host Disease

In long-term survivors of allogeneic hematopoietic cell transplantation (HCT), chronic graft-versus-host disease (GVHD) is the major cause of morbidity and mortality and a major determinant of quality of life. Chronic GVHD responds poorly to current immunosuppressive drugs, and while T cell depletion may be preventive, this gain is offset by increased relapse rates. A significant impediment to progress in treating chronic GVHD has been the limitations of existing animal models. The goal of this study was to develop a reproducible comprehensive model of chronic GVHD in the dog. Ten recipient dogs received 920 cGy total body irradiation, infusion of marrow, and an infusion of buffy coat cells from a dog leukocyte antigen (DLA)–mismatched unrelated donor. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, 11) and cyclosporine. The duration of cyclosporine administration was limited to 80 days instead of the clinically used 180 days. This was done to contain costs, as chronic GVHD was expected to develop at earlier time points. All recipients were given ursodiol for liver protection. One dog had graft failure and 9 dogs showed stable engraftment. Eight of the 9 developed de novo chronic GVHD. Dogs progressed with clinical signs of chronic GVHD over a period of 43 to 164 (median, 88) days after discontinuation of cyclosporine. Target organs showed the spectrum of chronic GVHD manifestations that are typically seen clinically. These included lichenoid changes of the skin, fasciitis, ocular involvement (xerophthalmia), conjunctivitis, bronchiolitis obliterans, salivary gland involvement, gingivitis, esophageal involvement, and hepatic involvement. Peripheral blood lymphocyte surface antigen expression of CD28 and inducible costimulator was elevated in dogs with GHVD compared with those in normal dogs, but not significantly so. Serum levels of IL-8 and monocyte chemotactic protein-1 in GVHD-affected dogs at time of euthanasia were elevated, whereas levels of IL-15 were depressed compared with those in normal dogs. Results indicate that the canine model is well suited for future studies aimed at preventing or treating chronic GVHD.

In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes.

Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil, a new immunosuppressive drug effective in the treatment of canine autoimmune diseases. The impact of MPA on immunity is ambiguous and its influence on the canine immune system is unknown. The aim of the study was to determine markers of changes in stimulated peripheral canine lymphocytes after treatment with MPA in vitro. Twenty nine healthy dogs were studied. Phenotypic and functional analysis of lymphocytes was performed on peripheral blood mononuclear cells cultured with mitogens and different MPA concentrations– 1 μM (10−3 mol/m3), 10 μM or 100 μM. Apoptotic cells were detected by Annexin V and 7-aminoactinomycin D (7-AAD). The expression of antigens (CD3, CD4, CD8, CD21, CD25, forkhead box P3 [FoxP3] and proliferating cell nuclear antigen [PCNA]) was assessed with monoclonal antibodies. The proliferation indices were analyzed in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells. All analyses were performed using flow cytometry. The influence of MPA on apoptosis was dependent on the mechanism of cell activation and MPA concentration. MPA caused a decrease in the expression of lymphocyte surface antigens, CD3, CD8 and CD25. Its impact on the expression of CD4 and CD21 was negligible. Its negative influence on the expression of FoxP3 was dependent on cell stimulation. MPA inhibited lymphocyte proliferation. In conclusion, MPA inhibited the activity of stimulated canine lymphocytes by blocking lymphocyte activation and proliferation. The influence of MPA on the development of immune tolerance–expansion of Treg cells and lymphocyte apoptosis–was ambiguous and was dependent on the mechanism of cellular activation. The concentration that MPA reaches in the blood may lead to inhibition of the functions of the canine immune system. The applied panel of markers can be used for evaluation of the effects of immunosuppressive compounds in the dog.

CYTOTOXIC LYMPHOCYTES: THE ROLE IN INFLAMMATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATION AND REMISSION

Background: Despite the number of publications related to the expression of surface antigens of periphery blood lymphocytes in chronic obstructive pulmonary disease (COPD), algorithm for interpreting of the results and implicating pathogenetic treatments still needs to be developed. Aim: To assess the role of cytotoxic lymphocytes in the maintaining of inflammation in COPD. Materials and methods: To examine immune status in 37 patients with COPD exacerbation or remission and 24 healthy donors (control group), blood cytotoxic T-lymphocytes and NK-cells contents were measured using indirect immunofluorescence method. Absolute and relative numbers of lymphocytes expressing CD3, CD4, CD8, CD16, CD20, CD23, CD25, CD54, CD71, CD72, HLA-DR, CD95 antigens, membrane immunoglobulins M (mIgM) and G (mIgG) were estimated. Results: In COPD., significantly increased numbers of blood cytotoxic lymphocytes were demonstrated independently from the disease stage (p < 0.001). During COPD exacerbation, significant elevations of CD4, CD8, CD20, CD72, NK-cells numbers, serum mIgM and mIgG were demonstrated. During remission, CD20 and CD72 content returned to normal, though, increased numbers of other cytotoxic cells persisted promoting inflammation and progressive damage of pulmonary and bronchial tissues. Conclusion: Observed changes may be due to excessive stimulation of T-cell component of immune system in COPD patients both in exacerbation and remission. Relative reduction of total T-lymphocyte numbers indicates non-specific (non-infectious) inflammation type. High cytotoxic potential of immune system results in pulmonary damage and promotes development of pneumosclerosis and emphysema.

Prospective Study of Biomarkers of Immune Response in Lung Transplant Recipients

IntroductionStudies on biomarkers of tolerance in organ transplantation have been widely performed during the last decade. AimTo assess biomarkers in relation to evolution of the immune response among lung transplant recipients. MethodsThis multicenter study included 27 lung transplant recipients followed before as well as at 7, 14, 30, 60, 90, and 180 days posttransplantation. Biomarkers of the immune response based on flow cytometry technology were validated in each center. They included intracellular cytokine expression, regulatory T-cell level, as well as lymphocyte surface antigen and CD28 expressions. ResultsThe 13 patients who developed acute rejection episodes showed increased numbers of regulatory T cells at 12 months posttransplant. Sixteen patients experiencing infections displayed decreased expression of CD69 on CD8 T cells within the first year of follow-up. ConclusionHigh Treg levels in the peripheral blood of lung transplant recipients were associated with an increased risk of rejection but not infection. Inversely, we observed low levels of activated CD8 T cells in infected patients.

Anti-oxidative and anti-inflammatory effects of spirulina on rat model of non-alcoholic steatohepatitis

The pathogenesis of nonalcoholic steatohepatitis (NASH) remains unclear, but accumulating data suggest oxidative stress and the relationship between inflammation and immunity plays a crucial role. The aim of this study is to investigate the spirulina, which is a blue-green algae rich in proteins and other nutritional elements, and its component-phycocyanin effect on a rat model of NASH. NASH model rats were established by feeding male Wistar rats with choline-deficient high-fat diet (CDHF) and intermittent hypoxemia by sodium nitrite challenge after 5 weeks of CDHF. After experimental period of 10 weeks, blood and liver were collected to determine oxidative stress injuries and efficacies of spirulina or phycocyanin on NASH model rats. In the NASH model rats, increase in plasma liver enzymes and liver fibrosis, increases in productions of reactive oxygen species from liver mitochondria and from leukocytes, the activation of nuclear factor-kappa B, and the change in the lymphocyte surface antigen ratio (CD4+/CD8+) were observed. The spirulina and phycocyanin administration significantly abated these changes. The spirulina or phycocyanin administration to model rats of NASH might lessen the inflammatory response through anti-oxidative and anti-inflammatory mechanisms, breaking the crosstalk between oxidative stress and inflammation, and effectively inhibit NASH progression.

A modified method for isolation of infiltrating lymphocytes in cardiac grafts

Objective To introduce a simple and efficient method for isolation of intragraft infiltrating lymphocytes. Methods Cardiac allografts were cut into small pieces that were then digested with collagenase Ⅱ (250 U/ml, 30-40 min). Mononuclear cells were isolated by Ficoll density gradient centrifugation (800 g/min, 20 min). Subsets of lymphocytes were identified by flow cytometry after surface antigen and intracellular cytokine staining. Results The harvested mononuclear cells were more than 1 × 106, which contained (31.9 ±2. 3)% lymphocytes with viability of (95.1 ±2. 1 )%. Flow cytometry could analyze surface and intracellular antigens of the lymphocytes at the same time. Conclusion The modified method of isolating intragraft infiltrating lymphocytes with high viability is simple, reliable and efficient. Key words: Cardiac allograft; Lymphocytes; Allograft; Collagenase Ⅱ; Flow cytometry

Expression of cytolytic protein–perforin in peripheral blood lymphocytes in severe traumatic brain injured patients

PurposeThe purpose of this study was to investigate the changes of cytotoxic protein–perforin in peripheral blood lymphocytes in severe TBI patients and possible correlation between severity of TBI and perforin expression. MethodsFlow cytometry was used for simultaneous detection of intracellular perforin and cell surface antigens of peripheral blood lymphocytes of 20 severe TBI patients on day 1, 4 and 7 after the onset of injury. Peripheral blood mononuclear cells from 20 healthy volunteers were used as control. Clinical and laboratory parameters were also recorded. ResultsThere was a statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells on day 4 as compared with day 1 after the brain injury or healthy controls. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8+ T lymphocytes, NK cells, and NKT cells) compared with day 1. High positive correlation was found between the severity of TBI and frequency of perforin-positive cells on day 4 when the occurrence of the intra-hospital infections was the highest. ConclusionSevere TBI significantly decreases perforin expression in T lymphocytes, NK and NKT cells, which indicate a possible mechanism underlying the high susceptibility to infections.

Rituximab for the treatment of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5(+)/CD19(+)/CD20(+)/HLA-DR+/CD23(+)/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival. The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.

Rituximab for the treatment of patients with chronic lymphocytic leukemia

Rituximab for the treatment of patients with chronic lymphocytic leukemia M Gentile, E Vigna, C Mazzone, E Lucia, AG Recchia, L Morabito2, MG Bisconte, C Gentile, F Morabito1UOC di Ematologia, Azienda Ospedaliera di Cosenza, Italy; 2Servicio de Hematolog&iacute;a y Hemoterapia, Hospital Universitario de Canarias, La Laguna, Tenerife, SpainAbstract: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5+/CD19+/CD20+/HLA-DR+/CD23+/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival . The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.Keywords: rituximab, chronic lymphocytic leukemia, chemotherapy