Evaluation of the Levels of Peripheral CD3+, CD4+, and CD8+ T Cells and IgG and IgM Antibodies in COVID-19 Patients at Different Stages of Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects the stimulatory levels of cellular-mediated immunity, which plays an essential role in controlling SARS-CoV-2 infection. In fact, several studies have shown the association of lymphopenia with severe COVID-19 in patients. The aim of this study is to investigate the response of the immune system, including cell-mediated immunity and antibody production, during different stages of SARS-CoV-2 infection. Peripheral blood and serum samples were collected from patients with moderate infection, patients under medication (hospitalized), patients who had recovered, and healthy individuals (n = 80). Flow cytometry analysis was performed on peripheral blood samples to determine the cellular immunity profile of each patient. The data showed a significant reduction in the levels of CD3+, CD4+, and CD8+ T cells and CD45+ cells in the moderate and under-medication groups, suggesting lymphopenia in those patients. Also, enzyme-linked immunosorbent assay (ELISA) was conducted on the serum samples to measure the levels of antibodies, including IgM and IgG, in each patient. The results revealed a significant increase in the levels of IgM in the moderate infection and under-medication patients, thus indicating the production of IgM during the first week of infection. Furthermore, changes in the levels of IgG were significantly detected among recovered patients, indicating therefore a remarkable increase during the recovery stage of SARS-CoV-2 infection and thus a strong humoral-mediated immunity. In summary, the results of this study may help us to understand the main role of the cellular immune responses, including CD3+, CD4+, and CD8+ T cells, against SARS-CoV-2 infection. This understanding might support the development of SARS-CoV-2 treatments and vaccines in the near future. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 in China. This virus is a serious threat to people not only in China but also worldwide, where it has been detected in over 222 countries. It has been reported that ∼3.4% of SARS-CoV-2-infected patients have died. The significance of our study relies on the fact that an enzyme-linked immunosorbent assay and flow cytometry were used to measure the levels of antibodies and cellular immune response, respectively, from clinical samples of patients infected with SARS-CoV-2.

CD3+T, CD4+T, CD8+T, and CD4+T/CD8+T Ratio and Quantity of γδT Cells in Peripheral Blood of HIV-Infected/AIDS Patients and Its Clinical Significance.

OBJECTIVE: To investigate the quantity of CD4+T, CD4+T, CD8+T, and γδT cells in peripheral blood of HIV-infected/AIDS patients as well as to explore the possible role of CD4/CD8 ratio and γδT cells in the progression of HIV/AIDS, aimed at providing evidence for the diagnosis and treatment of AIDS. METHODS: The quantity levels of CD3+T cells, CD4+T cells, CD8+T cells, and γδT cells in peripheral blood of 46 HIV-infected/AIDS patients and 30 healthy controls were detected by using flow cytometry. RESULTS: The count of CD3+T, CD4+T, CD8+T, and γδT cells ( x ¯ ± s , A/µl) in the peripheral blood was 1183.64 ± 132.58, 278.39 ± 122.38, 863.13 ± 82.38, and 22.53 ± 1.74 in the experimental group as well as 1456.46 ± 124.37, 788.74 ± 189.67, 569.61 ± 46.49, and 10.96 ± 0.28 in the control group, respectively. The p values of the two groups were <0.005 after the t-test, revealing a statistically significant difference. The proportion of CD3+T, CD4+T, CD8+T, and γδT cells in total lymphocytes in the two groups ( x ¯ ± s , %) was 71.83 ± 5.37, 13.39 ± 2.23, 62.93 ± 5.81, and 3.67 ± 0.87 in the experimental group, respectively. In the control group, the values were expressed as 66.72 ± 5.48, 42.77 ± 3.38, 31.41 ± 3.62, and 1.73 ± 0.36, respectively. After performing the t-test, p values in the two groups were <0.005 except CD3+T, with statistically significant differences. Besides, CD4/CD8 was 0.33 ± 0.11 in the experimental group and 1.48 ± 0.29 in the control group, t = 26.528, p < 0.001, exhibiting a significant statistical difference. CONCLUSION: HIV infection induces the activation and proliferation of CD8+T and γδT cells, contributing to the decrease of CD4+T cells, while CD8+T and γδT cells are involved in the immune response and tissue damage after HIV infection.

CD247, a Potential T Cell-Derived Disease Severity and Prognostic Biomarker in Patients With Idiopathic Pulmonary Fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear. Methods: Datasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis. Results: CD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein-protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils. Conclusion: These results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF.

Biomarkers involved in evaluation of platelets function in South-Eastern Romanian patients with hematological malignancies subtypes.

ABSTRACT: At present, various researches presented how subtypes of hematological malignancies are related to stages of the immune response, because the activated immune system represents a promising form in cancer treatment. This study explores the relationship between the adaptive immune system (T cells), and the coagulation system (platelets, platelet membrane glycoproteins, platelets derivate microparticles) which seems to play an important role in host immune defense of patients with acute myeloblastic leukemia (AML) or B cell lymphoma (BCL), 2 of the most common hematological malignancies subtypes.Blood samples (n = 114) obtained from patients with AML or BCL were analyzed for platelet membrane glycoproteins (CD42b, CD61), glycoprotein found on the surface of the T helper cells (CD4+), protein complex-specific antigen for T cells (CD3+), platelet-derived microparticles (CD61 PMP) biomarkers by flow cytometry, and hematological parameters were quantified by usual methods.In patients with AML, the means of the percentage of the expressions of the molecules on platelet surfaces (CD61 and CD42b, P < .01; paired T test) were lower as compared to both control subgroups. The expression of cytoplasmic granules content (CD61 PMP) had a significantly higher value in patients with AML reported to controlling subgroups (P < .01; paired T test), which is suggesting an intravascular activation of platelets.The platelet activation status was presented in patients with low stage BCL because CD61 and CD42b expressions were significantly higher than control subgroups, but the expression of CD 61 PMP had a significantly decreased value reported to control subgroups (all P < .01; paired T test). T helper/inducer lineage CD4+ and T lymphoid lineage CD3+ expressions presented significant differences between patients with AML or low stage BCL reported to control subgroups (all P < .01; paired T test).Platelet-lymphocyte interactions are involved in malignant disorders, and CD61, CD42b present on platelet membranes, as functionally active surface receptors mediate the adhesion of active platelets to lymphocytes, endothelial cells, and cancer cells.

Blood type change identifies late dominance reversal of chimerism after double umbilical cord blood transplantation with review of the literature.

BACKGROUND: A 30-year-old man underwent double umbilical cord blood transplantation (UCBT) for acute myeloid leukemia (AML) with reduced intensity conditioning. The cords had identical HLA types and were each a 5/6 match to the patient. Following transplantation, cord 2 initially dominated all tested cell populations. At day +306, we observed an unusual reversal of dominance chimerism pattern in which cord 1 instead dominated all tested populations. STUDY DESIGN & METHODS: Polymerase chain reaction (PCR)-based short tandem repeat (STR) assays were performed on the peripheral blood and bone marrow samples. The white blood cell (WBC) populations from the peripheral blood were manipulated for testing to create subpopulations enriched for CD3, CD33, and CD56. RESULTS: Chimerism studies on day +77 showed the following: cord 1: 44%-CD3; 0%-CD33; 16%-CD56; cord 2: 56%-CD3; 100%-CD33; 84%-CD56. Cord 2 initially dominated in all tested cell populations. Chimerism studies performed on post-transplantation day +306 uncovered a reversal of dominance chimerism pattern in which cord 1 now dominated in all cell populations (cord 1: 82%-CD3; >95%-CD33; 67%-CD56; cord 2: 18%-CD3; <5%-CD33; 33%-CD56). Between days +127 and +244, the patient's blood type shifted from B Rh-positive to A Rh-negative. CONCLUSION: The change in the patient's blood type identified a late reversal of dominance chimerism pattern. This is a rare occurrence, previously cited only once, which is inconsistent with published data that early high CD3 counts and unseparated bone marrow chimerism predominance at day +100 predict long-term cord dominance in double UCBT in the vast majority of cases.

Serum levels of the IL-6 family of cytokines predict prognosis in renal cell carcinoma (RCC).

PURPOSE: An improved understanding of RCC immunology should shed further light on RCC tumor biology. Our objective was to study to what extent serum levels of the IL-6 family of cytokines at diagnosis were relevant to survival. METHODS: A total of 118 consecutively patients with RCC, in which the tumor was surgically removed at Haukeland University Hospital during the period from 2007 to 2010, were included. The patients were followed-up for 10 years. The morning before surgery blood was sampled and serum frozen, with levels of IL-6, IL-27, IL-31, OSM, CNTF, IL-6Rα and gp130 determined. RESULTS: Among patients with the highest quartile of IL-6 (> 8 pg/ml) (n = 29), six of nine who had metastasis at diagnosis had such high IL-6 values. Among presumed radically treated patients, a high IL-6 and IL-27 strongly predicted recurrence. In particular, the predictions among patients with large (diameter > 7 cm) tumors were excellent regarding both IL-6 and IL-27 values. High gp130 serum levels predicted an overall survival (OS) among RCC patients with large tumors. Patients with a high IL-6 exhibited a strong expression of IL-6 in endothelial- and vascular smooth muscle cells. Moreover, the level of intra-tumoral CD3-positive cells predicted survival. CONCLUSIONS: IL-6 and IL-27 seem to play a role in RCC biology. IL-6 enables the pinpointing of metastatic condition at diagnosis, as well as together with IL-27, the predicting of survival and recurrence. Endothelial cells and vascular smooth muscle cells are both suggested as important sources of IL-6.

Primary pulmonary extranodal natural killer/T-cell lymphoma (ENKTL), nasal type: Two case reports and literature review.

INTRODUCTION: Extranodal natural killer/T-cell lymphoma (ENKTL) - nasal type is an aggressive form of malignant non-Hodgkin lymphoma with a very poor prognosis. Especially primary pulmonary ENKTL is a relatively rare form of non-Hodgkin lymphoma. Until now, the prevalence of primary pulmonary ENKTL is unknown. Since 2001, only 18 cases of primary pulmonary ENKTL have been published, in addition to the 2 cases reported here. PATIENT CONCERNS: We describe 2 cases of primary pulmonary ENKTL. Both patients were male non-smokers, aged 61 and 49 years. Their main clinical symptoms included cold-like symptoms and intermittent fever (39.3°C and 38.8°C) for some days (40 days and 3 weeks). Both patients had no relevant personal or family medical history. DIAGNOSIS: The patients were initially misdiagnosed with community-acquired pneumonia. Primary pulmonary ENKTL was confirmed by immunohistochemical staining of computed tomography-guided transthoracic needle biopsy specimens. Both cases were positive for CD56, CD3, and in situ hybridization for Epstein-Barr virus-encoded small RNA, but negative for CD20. INTERVENTIONS: Initially, both patients were treated inadequately with intravenous moxifloxacin administration (unknown dosage and 400 mg q.d) in their local hospitals. Once diagnosed with primary pulmonary ENKTL in our hospital, they received 3 cycles of chemotherapy with combined regimens of dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide (SMILE), and in the second patient, bone marrow transplantation was performed following the third chemotherapy cycle. OUTCOMES: Clinical follow-up after the chemotherapy showed that the condition of the first patient progressively deteriorated. He died 2 months following the initial diagnosis. However, the presence of the hemophagocytic lymphohistocytosis gradually improved in the second patient during chemotherapy. Ultimately, the second patient died of acute transplant rejection 6 months after the initial diagnosis. CONCLUSION: The diagnosis of ENKTL should be considered when patients present with fever and expansile consolidation of the lung not responding to antibiotics. The diagnosis depends on histopathology and immunophenotyping. Percutaneous transthoracic needle biopsy is a safe and effective biopsy method. Chemotherapy may improve the prognosis, but this should be confirmed by prospective multicenter studies.

Combination of four clinical indicators predicts the severe/critical symptom of patients infected COVID-19.

BACKGROUND: Despite the death rate of COVID-19 is less than 3%, the fatality rate of severe/critical cases is high, according to World Health Organization (WHO). Thus, screening the severe/critical cases before symptom occurs effectively saves medical resources. METHODS AND MATERIALS: In this study, all 336 cases of patients infected COVID-19 in Shanghai to March 12th, were retrospectively enrolled, and divided in to training and test datasets. In addition, 220 clinical and laboratory observations/records were also collected. Clinical indicators were associated with severe/critical symptoms were identified and a model for severe/critical symptom prediction was developed. RESULTS: Totally, 36 clinical indicators significantly associated with severe/critical symptom were identified. The clinical indicators are mainly thyroxine, immune related cells and products. Support Vector Machine (SVM) and optimized combination of age, GSH, CD3 ratio and total protein has a good performance in discriminating the mild and severe/critical cases. The area under receiving operating curve (AUROC) reached 0.9996 and 0.9757 in the training and testing dataset, respectively. When the using cut-off value as 0.0667, the recall rate was 93.33 % and 100 % in the training and testing datasets, separately. Cox multivariate regression and survival analyses revealed that the model significantly discriminated the severe/critical cases and used the information of the selected clinical indicators. CONCLUSION: The model was robust and effective in predicting the severe/critical COVID cases.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Absence of influence of peripheral blood CD34+ and CD3+ graft cell counts on outcomes after reduced-intensity conditioning transplantation using post-transplant cyclophosphamide.

The influence of peripheral blood stem cell (PBSC) graft cell contents after transplant with post-transplant cyclophosphamide (PTCY) remains unclear. Here, we retrospectively report on a cohort of 77 adults who received a Baltimore-based reduced-intensity conditioning regimen either with fludarabine (n = 40) or clofarabine (n = 37) and PTCY. With a median follow-up of 29.2 months, [2-]year overall (OS), disease-free (DFS), and GVHD/relapse-free survival (GRFS) rates were 62.8%, 51%, and 36.7%, respectively. The incidence of grades [2-]4 acute GVHD was significantly higher in patients transplanted with a haplodonor (n = 56), at 57.1% vs 19% (p = 0.006). PBSC graft cell contents (CD45+, CD34+, and CD3+ cells) had no impact on any outcome. Considering immune reconstitution until 1 year, only monocytes were above the normal range (as early as day + 30) during the first year post-transplant. In multivariate analysis, an older donor (> 45 years) and a high/very high disease risk index were independently associated with lower OS. A higher monocyte count (> median) at day + 90 was also associated with better OS, DFS, and GRFS. Donor/recipient CMV status matching was independently associated with GRFS. In conclusion, our data support the fact that there is no need to manipulate the graft before infusion in the particular context of PBSC/PTCY Baltimore-based allotransplant.

Characterization of the immune cell landscape of patients with NAFLD.

Multiple factors are involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), but the exact immunological mechanisms that cause inflammation and fibrosis of the liver remain enigmatic. In this current study, cellular samples of a cohort of NAFLD patients (peripheral blood mononuclear cells (PBMC): n = 27, liver samples: n = 15) and healthy individuals (PBMC: n = 26, liver samples: n = 3) were analyzed using 16-color flow cytometry, and the frequency and phenotype of 23 immune cell subtypes was assessed. PBMC of NAFLD patients showed decreased frequencies of total CD3+, CD8+ T cells, CD56dim NK cells and MAIT cells, but elevated frequencies of CD4+ T cells and Th2 cells compared to healthy controls. Intrahepatic lymphocytes (IHL) of NAFLD patients showed decreased frequencies of total T cells, total CD8+ T cells, Vd2+γδ T cells, and CD56bright NK cells, but elevated frequencies of Vδ2-γδ T cells and CD56dim NK cells compared to healthy controls. The activating receptor NKG2D was significantly less frequently expressed among iNKT cells, total NK cells and CD56dim NK cells of PBMC of NAFLD patients compared to healthy controls. More strikingly, hepatic fibrosis as measured by fibroscan elastography negatively correlated with the intrahepatic frequency of total NK cells (r2 = 0,3737, p = 0,02). Hepatic steatosis as measured by controlled attenuation parameter (CAP) value negatively correlated with the frequency of circulating NKG2D+ iNKT cells (r2 = 0,3365, p = 0,0047). Our data provide an overview of the circulating and intrahepatic immune cell composition of NAFLD patients, and point towards a potential role of NK cells and iNKT cells for the regulation of hepatic fibrosis and steatosis in NAFLD.

Differences in the relative counts of peripheral blood lymphocyte subsets in various age groups of pigs.

The aim of the study was to determine age-related changes in peripheral blood lymphocytes in pigs. Previous studies looking at age-related differences in lymphocyte subsets in porcine blood have not established reference ranges for these parameters. Moreover, most studies have concentrated on the dynamic changes in the first months of life, failing to continue observations in older animals. Therefore, in the present study, relative counts of various lymphocyte subpopulations (cytotoxic and helper T-cells, B-cells, and γδ T-cells) were evaluated to characterize the development of the cellular immune system at 28, 35, 135, and 200 days of age in growing pigs and adult sows (i.e., first and subsequent parity). In all examined groups, CD3+ cells constituted the largest percentage of cells. A statistically significant higher percentage of TCRγδ+CD3+ was noted in fatteners and gilts in comparison to other age groups. These results may be a reflection of antigenic pressure and show an immune response to viral or bacterial agents/environmental microbism.

L'objectif de la présente étude était de déterminer les changements associés à l'âge dans les lymphocytes du sang périphérique chez les porcs. Des études antérieures examinant les différences associées à l'âge dans les sous-populations de lymphocytes dans le sang porcin n'ont pas établi des écarts de référence pour ces paramètres. De plus, la plupart des études se sont concentrées sur les changements dynamiques dans les premiers mois de vie, omettant de continuer les observations chez les animaux plus âgés. Ainsi, dans la présente étude, les dénombrements relatifs des différentes sous-populations lymphocytaires (cellules-T cytotoxiques et helper, cellules-B, et cellules-T γδ) furent évalués afin de caractériser le développement du système immunitaire cellulaire à 28, 35, 135, et 200 jours d'âge chez des porcs en croissance et des truies adultes (i.e. première parité ainsi que les suivantes). Dans tous les groupes examinés, les cellules CD3+ constituaient le pourcentage le plus élevé de cellules. Un pourcentage significativement plus élevé de TCRγδ+CD3+ était noté chez les porcs en croissance et les cochettes comparativement aux autres groupes d'âge. Ces résultats pourraient être un reflet de la pression antigénique et montre une réponse immunitaire à des agents viraux ou bactériens du microbisme environnemental.(Traduit par Docteur Serge Messier).

Ki-67/CD3 ratio in the diagnosis of chronic inflammatory enteropathy in dogs.

BACKGROUND: T cells play a key role in the pathogenesis of chronic inflammatory enteropathy (CIE) in dogs. Cluster of differentiation 3 (CD3) antigen serves as a marker for T cells. In human medicine, Ki-67 is an indicator for cell growth but there are only a few studies in dogs with CIE. OBJECTIVE: To investigate Ki-67 in relation to T cells as a marker for CIE in dogs. ANIMALS: Eleven dogs with CIE and 6 healthy beagle controls (CO). METHODS: Retrospective case-control study. Dogs were clinically assessed by the Canine Chronic Enteropathy Clinical Activity Index (CCECAI). Duodenal mucosal biopsy samples were endoscopically obtained for histopathologic examination by means of the World Small Animal Veterinary Association score. Double-labeled immunofluorescence was used to investigate colocalization of Ki-67 and CD3 in epithelium and lamina propria (LP) of villi and crypts. RESULTS: Dogs with CIE had significantly higher clinical score (median, 5.0; interquartile range [IQR], 3-7) compared to CO (all 0; P < .001). The Ki-67/CD3 double-positive cells were significantly increased in the LP of the crypt region of CIE dogs (0.63 cells/mm2 ; IQR, 0-0.54) versus CO (0.08 cells/mm2 ; IQR, 0-0.26; P = .044). A significant correlation was found between CCECAI and the Ki-67/CD3 ratio in the LP of the crypt region (r = 0.670; P = .012) in dogs with CIE. CONCLUSIONS AND CLINICAL IMPORTANCE: The Ki-67/CD3 ratio is upregulated in the LP crypt region of dogs with CIE and it correlates with clinical severity. Therefore, Ki-67/CD3 could be a useful tool for detection of CIE.

Validation of simple prediction algorithms to consistently achieve CD3+ and postselection CD34+ targets with leukapheresis.

BACKGROUND: Cellular therapies using engineered T cells, haploidentical transplants, and autologous gene therapy are increasing. Specified CD3+ or high CD34+ doses are typically required for subsequent manufacturing, manipulation, or CD34+ selection. Simple, practical, and reliable lymphocyte and hematopoietic progenitor cell (HPC) collection algorithms accounting for subsequent CD34+ selection have not been published. STUDY DESIGN AND METHODS: In this analysis of 15 haploidentical donors undergoing tandem lymphocyte and HPC collections, we validated one-step, practical prediction algorithms (Appendix S1, available as supporting information in the online version of this paper) that use conservative facility-specific collection efficiencies, CD34+ selection efficiency, and donor-specific peripheral counts to reliably achieve the target CD3+ and CD34+ product doses. These algorithms expand on our previously published work regarding predictive HPC collection algorithms. RESULTS: Ninety-three percent of lymphocyte and 93% of CD34+ collections achieved the final target CD3+ and CD34+ product dose when our algorithm-calculated process volumes were used. Linear regression analysis of our algorithms for CD3+, preselection CD34+, and postselection CD34+ showed statistically significant models with R2 of 0.80 (root mean square error [RMSE], 31.3), 0.72 (RMSE, 385.7), and 0.56 (RMSE, 326.0), respectively, all with p values less than 0.001. CONCLUSION: Because achievement of CD3+ or CD34+ dose targets may be critical for safety and efficacy of cell therapies, these simple, practical, and reliable prediction algorithms for lymphocyte and HPC collections should be very useful for collection facilities.

Blood lymphocyte T subsets reference values in blood donors by flow cytometry.

AIMS: To determine region-specific reference ranges of lymphocyte T subsets in blood donors and to assess the influence of gender and age on lymphocyte T susbsets. METHODS: Blood samples from 143 blood donors were collected in the Blood Transfusion Center of Sfax. Lymphocyte T subsets were analyzed using a dual-platform method with a flow cytometer (percentages) and an automated hematology analyzer (white blood cells and lymphocytes). ANOVA and Student's tests were used for data analysis. RESULTS: Reference values were expressed as mean and 95% confidence intervals for T cells: CD3+: 1415 ± 348 cells/µL [1357-1473], CD3+/CD4+: 786 ± 220 cells/µL [732.31-811.7], CD3+/CD8+: 639 ± 258 cells/µL [596-862] and CD4+/CD8+ ratio was 1.46 ± 0.77 [1.36-1.62]. Gender and age influenced blood lymphocyte T subsets. CONCLUSION: Our study leads to the establishment of peripheral blood lymphocyte T subset reference ranges in blood donors in the region of Sfax. A study on a more diversified population, including more important number of individuals from various regions of Tunisia and including enumeration of other lymphocyte subsets (B cells and NK cells) is required.

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.

CD3, CD4, CD8, CD19 and CD16/CD56 positive cells in tuberculosis infection and disease: Peculiar features in children.

Pathogenesis of mycobacterial infection has been extensively studied determining the fundamental role of host immunocompetence in disease progression. Cellular adaptive immunity, in particular CD4+ cells, has shown to be crucial in the host defence. A role of cytotoxic lymphocytes and humoral immunity has also been established. However, few studies have been performed in low endemic countries on immunological correlates of tuberculosis in paediatric patients. The present study aims to fill this gap analysing the distribution and the absolute values of the main lymphocyte subpopulations (CD3+, CD4+, CD8+, CD19+ and CD16+/CD56+) in the different stages of tubercular infection in human immunodeficiency virus-negative children living in low tubercular endemic countries. Results obtained in children with latent tuberculosis, active tuberculosis and healthy controls were compared. Moreover, quantitative analysis of interferon-γ levels of mitogen-induced response was carried out within the different study groups. The aim of this analysis was to enforce the comprehension of immune modifications subsequent to Mycobacterium tuberculosis infection. The major finding of our study was CD3+ and CD4+ absolute and percentage depletion in children with active tuberculosis versus healthy controls. Moreover, severe forms of active tuberculosis showed a marked reduction in the CD4+ percentage in the context of a systemic impairment which affects globally the absolute count of all peripheral lymphocyte subsets tested. A relative increase of natural killer cells was proved in infected patients, whereas no differences in B cells among the study groups were detected. Mitogen-induced interferon-γ levels were significantly higher in children with latent tuberculosis when compared to active tuberculosis and healthy controls, demonstrating effective immune activation in those patients able to control the infection.

Predictive value of combined serum FGF21 and free T3 for survival in septic patients.

BACKGROUND: We examined the correlation between thyroid hormone (TH) concentrations and the serum fibroblast growth factor 21 (FGF21) concentration in septic patients and to assess the collaborative value of these factors in predicting 28-day mortality in septic patients. METHODS: A total of 120 consecutive patients with sepsis were divided into two groups according to their survival or death within 28 days after initial diagnosis of sepsis. RESULTS: Patients in the non-survivor group had significantly higher serum FGF21 concentrations but lower total and free triiodothyronine (T3) and tetraiodothyronine (T4) concentrations than those in the survivor group. Thyroid hormone concentrations, including T3, free T3, T4 and free T4, were significantly negatively correlated with the ∆SOFA and APACHE II scores as well as the serum FGF21, IL-6, tumor necrosis factor-α, IL-10, procalcitonin, and C-reactive protein concentrations. Logistic regression analysis showed that the ∆SOFA score, serum FGF21 concentration, and free T3 concentration were significant predictors of 28-day mortality. The model with variables of ∆SOFA score and serum FGF21 and free T3 concentrations had the greatest area under the curve of 0.969. CONCLUSION: The addition of free T3 and serum FGF21 to ∆SOFA score provided a significantly improved ability to predict 28-day mortality in septic patients.