Higher Proportion Of Lymphocytes Research Articles

Abstract TMP89: Thrombus Cellular Composition Predicts Unexpected Early Intracranial Reocclusion After Successful Mechanical Thrombectomy

Introduction: Our objective is to identify thrombus composition and related factors associated with unexpected early intracranial reoclussion ( EIR) after successful mechanical thrombectomy ( MT). Methods: Endovascular treatments of patients with acute intracranial or extracranial stenting were excluded. A total of 206 patients with successful reperfusion ( mTICI ≥ 2b) were included . Each intracranial thrombi obtained by MT were analyzed by Flow Cytometry analysis to provide proportion of main leukocyte composition: granulocytes, monocytes and lymphocyte populations. Thrombi analysis, antithrombotic treatment, etiology , reperfusion treatment features, neuroimaging and other baseline variables were analyzed to identify predictors of early reoclussion in the first 48 hours after thrombectomy . Results: EIT were observed in 5.8 %( 12/206) of successfully reperfused strokes. Patients with EIR were younger [ 67 years ( 47-80) vs. 76 years ( 66-84) ; p = 0.046] with lower basal NIHSS [ 17 ( 10-21) vs. 18 ( 11-21) ; p = 0.022]. Thrombi from patients with EIR had higher proportion of lymphocytes B [1.41 % ( 0.48-2.42) vs. 0.44 %(0.16-1.19) ;p = 0.018] in comparison to cases without reoclussion. Basilar artery occlusion ( BAO) [ 2 (15.4%) vs. 8 (4,3%) ; p 0.081 ] and active smoking [ 4( 30.8%) vs. 24 (12.4%) p =0.062] presented a positive trend with EIR cases . In the logistic regression analysis adjusted by age, previous use of intravenous fibrinolysis, number of endovascular passes , proportion of Lymphocytes B, BAO and active smoking , the independent predictor of early reoclussion was the proportion of Lymphocyte B in intracranial thrombus ( aOR 1.363 , 95% CI 1.060- 1.753). Conclusions: Rich B-Lymphocyte- Thrombus predicts unexpected early intracranial reoclusion after successful mechanical thrombectomy

Optimising the yield from bronchoalveolar lavage on human participants in infectious disease immunology research

Bronchoalveolar lavage (BAL) is becoming a common procedure for research into infectious disease immunology. Little is known about the clinical factors which influence the main outcomes of the procedure. In research participants who underwent BAL according to guidelines, the BAL volume yield, and cell yield, concentration, viability, pellet colour and differential count were analysed for association with important participant characteristics such as active tuberculosis (TB) disease, TB exposure, HIV infection and recent SARS-CoV-2 infection. In 337 participants, BAL volume and BAL cell count were correlated in those with active TB disease, and current smokers. The right middle lobe yielded the highest volume. BAL cell and volume yields were lower in older participants, who also had more neutrophils. Current smokers yielded lower volumes and higher numbers of all cell types, and usually had a black pellet. Active TB disease was associated with higher cell yields, but this declined at the end of treatment. HIV infection was associated with more bloody pellets, and recent SARS-CoV-2 infection with a higher proportion of lymphocytes. These results allow researchers to optimise their participant and end assay selection for projects involving lung immune cells.

Landscape of alterations in the checkpoint system in myelodysplastic syndrome and implications for prognosis.

The emergence of novel immunotherapies for myelodysplastic syndrome (MDS) calls for a profound characterization of the "immunome" in the bone marrow (BM) and evaluation of prognostic impact of immunological changes. We performed a prospective study of 87 MDS patients who were referred to a tertiary hematological center and of 11 bone marrow donors who were not related to the study cohort. A flow cytometry panel with 48 markers including checkpoint ligands and receptors was used to study lymphoid and myeloid subpopulations in the bone marrow aspirates. The study found that both the healthy donors and the MDS patients have a high proportion of lymphocytes with PD-1 expression (41±18% and 58±25% respectively) and a high proportion of myeloid cells with PD-1L expression (31±23% and 12±7% respectively), indicating a potential physiological role of checkpoint systems in BM. At the same time, complex alterations including PD-1, CTLA-4, LAG-3 and TIM3 pathways accompanied by an increased level of T-reg and myeloid derived suppressor cell populations were identified in the BM of MDS patients. Cluster analysis showed independent prognostic significance of the checkpoint profile for overall survival (HR 1.90, 95%CI 1.01–3.56, p = 0.0471). TIM3-postive NK and CD8 effector cells along with the blast count were the key subpopulations for prognosis. An elevation of blasts in the bone marrow was associated with simultaneous expression of multiple checkpoints on myeloid cells.

Immunological Status of Bladder Cancer Patients Based on Urine Leukocyte Composition at Radical Cystectomy.

Background: Bladder cancer (BC) is the ninth most common malignancy worldwide, with high rates of recurrence. The use of urine leukocyte composition at the time of radical cystectomy (RC) as a marker for the study of patients’ immunological status and to predict the recurrence of muscle-invasive bladder cancer (MIBC) has received little attention. Methods: Urine and matched peripheral blood samples were collected from 24 MIBC patients at the time of RC. Leukocyte composition and expression of PD-L1 and PD-1 in each subpopulation were determined by flow cytometry. Results: All MIBC patients had leukocytes in urine. There were different proportions of leukocyte subpopulations. The expression of PD-L1 and PD-1 on each subpopulation differed between patients. Neoadjuvant chemotherapy (NAC), smoking status, and the affectation of lymph nodes influenced urine composition. We observed a link between leukocytes in urine and blood circulation. Recurrent patients without NAC and with no affectation of lymph nodes had a higher proportion of lymphocytes, macrophages, and PD-L1+ neutrophils in urine than non-recurrent patients. Conclusions: Urine leukocyte composition may be a useful tool for analyzing the immunological status of MIBC patients. Urine cellular composition allowed us to identify a new subgroup of LN− patients with a higher risk of recurrence.

DNA Methylation-Based Estimates of Circulating Leukocyte Composition for Predicting Colorectal Cancer Survival: A Prospective Cohort Study

Simple SummaryInflammation is involved in the evolution of cancer. Leukocytes, of which the proportion can be estimated using epigenome-wide methylation data, may serve as a prognostic marker in colorectal cancer (CRC). Our aim was to investigate whether DNA methylation-based estimates of circulating leukocytes is associated with all-cause and disease-specific mortality in a prospective CRC patients’ cohort. Significant associations with CRC prognosis were observed for CD4+ T cells, CD8+ T cells, B cells, NK cells, and lymphocytes, independent of age, sex, tumor stage, tumor subsite, and therapy. CD4+ T cells outperformed other leukocytes and provided added predictive value in comparison to age, sex, and tumor stage. Although cell counting is commonly used in clinical practice, DNA methylation-estimated cell proportions could be a promising tool in understanding the role of leukocytes as CRC prognostic biomarkers when using stored blood samples.Leukocytes are involved in the progression of colorectal cancer (CRC). The proportion of six major leukocyte subtypes can be estimated using epigenome-wide DNA methylation (DNAm) data from stored blood samples. Whether the composition of circulating leukocytes can be used as a prognostic factor is unclear. DNAm-based leukocyte proportions were obtained from a prospective cohort of 2206 CRC patients. Multivariate Cox regression models and survival curves were applied to assess associations between leukocyte composition and survival outcomes. A higher proportion of lymphocytes, including CD4+ T cells, CD8+ T cells, B cells, and NK cells, was associated with better survival, while a higher proportion of neutrophils was associated with poorer survival. CD4+ T cells outperformed other leukocytes in estimating the patients’ prognosis. Comparing the highest quantile to the lowest quantile of CD4+ T cells, hazard ratios (95% confidence intervals) of all-cause and CRC-specific mortality were 0.59 (0.48, 0.72) and 0.59 (0.45, 0.77), respectively. Furthermore, the association of CD4+ T cells and prognosis was stronger among patients with early or intermediate CRC or patients with colon cancer. In conclusion, the composition of circulating leukocytes estimated from DNAm, particularly the proportions of CD4+ T cells, could be used as promising independent predictors of CRC survival.

The impact of parturition induction with cloprostenol on immunological parameters in the sow colostrum

BackgroundFarrowing induction with prostaglandin F2 analogue cloprostenol is commonly used on commercial farms to manage the timing of farrowing. When labour induction is applied, the questions arise about possible side effects of such a hormonal intervention on physiological processes connected with labour and lactation, including colostral immunity.ResultsIn this study, immune cells composition, lysozyme concentration, complement bacteriolytic activity and proinflamatory (GM-CSF2, IL-1β, IL-6, a TNFα) and anti-inflammatory (IL-4, IL-10, TGFβ1 a TGFβ2) cytokines were measured in colostrum samples from sows farrowing naturally (NP) and from sows with farrowing induced using cloprostenol administration on day 113 of gestation (IP). A significantly higher proportion of lymphocytes was found in colostrum of induced sows compared to colostrum of non-induced sows. No significant differences between NP and IP were found in complement activity, in the proportions of granulocytes, macrophages and lymphocyte subpopulations. Lower lysozyme concentration and higher IL-1β, IL-6, TGFβ1 and TNFα concentrations were found in IP sow colostrum compared to colostrum from NP sows.ConclusionsAn increased proportion of colostral lymphocytes can positively influence the cellular immunity transmission from sow to her offspring. On the other hand, a lower lysozyme concentration can adversely affect newborn’s intestinal immunity, as well as changes in cytokine concentrations can have an adverse effect on newborn piglet intestinal epithelium development and its defence function.

Coexistence of diffuse panbronchiolitis and sarcoidosis revealed during splenectomy: a case report

BackgroundSarcoidosis is a systemic granulomatous disease caused by CD4+ cell-dominant inflammation. Meanwhile, diffuse panbronchiolitis is a chronic inflammatory respiratory disease predominantly caused by CD8+ lymphocytes and neutrophils. Herein, we report a rare case of sarcoidosis in which the clinical presentation had become evident as diffuse panbronchiolitis after splenectomy for sarcoidosis.Case presentationA 23-year-old Japanese woman was referred to our hospital due to splenomegaly of unknown etiology. Upon admission, chest computed tomography scan revealed centrilobular and randomly distributed small nodules in both lungs. Bronchoalveolar lavage revealed a high proportion of lymphocytes and a decreased CD4/CD8 ratio. However, the biopsy specimens obtained from both the liver and lungs revealed noncaseating epithelioid granulomas, which confirmed the diagnosis of sarcoidosis. The patient underwent splenectomy due to progressive cytopenia and high risk of splenic rupture. After the surgery, the condition of the patient was consistently good for 3 months. Then, she gradually developed productive cough and dyspnea. Both sinus and chest computed tomography scan revealed chronic paranasal sinusitis and deterioration of centrilobular nodules in both lung fields, respectively. The second bronchoalveolar lavage revealed a high proportion of neutrophils, and the bronchoalveolar lavage fluid tested positive for Hemophilus influenzae. The titer of cold agglutinin was elevated, thereby confirming the diagnosis of diffuse panbronchiolitis. On the basis of the clinical and radiological findings, the condition of the patient improved with low-dose macrolide therapy for 3 months.ConclusionsThe coexistence of sarcoidosis and diffuse panbronchiolitis has not been previously reported, and the hidden profiles of diffuse panbronchiolitis may have been revealed by splenectomy.

Parental education associated with immune function in adolescence.

The immune system is affected and shaped by several internal and external factors. Among the external variables, the socioeconomic status is known to influence the immune system since the early years of life and throughout life. In this study, we assessed the relationship between parental education with the white blood cells and its subtypes in 1213 adolescents from the EPITeen cohort, assessed at the age of 13. Beta coefficients (β) and 95% confidence intervals (CI) were fitted using linear regression models to quantify the association and were adjusted for sex, body mass index and chronic disease. After adjustment, parental education presented a negative association with white blood cells, which was significant among those with higher high-sensitivity C-reactive protein (hs-CRP) median levels [-0.05mg/l (95% CI -0.08, -0.01)]. On the contrary, a positive association with lymphocytes was observed, which was, significant among those with lower hs-CRP [0.17mg/l (95% CI 0.02, 0.32)]. A neutrophil-to-lymphocyte ratio significant decrease was also observed with the increment of parental education (P<0.001). We found that parental education was positively associated with a higher proportion of lymphocytes and a lower proportion of neutrophils, suggesting that parental education is associated with offsprinǵs innate immune system regulation. These results may contribute to clarify the relationships between childhood socioeconomic status and increased risk of adverse cardiovascular outcomes and other immune-related diseases.

Personalized Peptide Vaccination for Castration-Resistant Prostate Cancer Progressing after Docetaxel Chemotherapy: A Randomised, Double-Blind, Placebo-Controlled, Phase 3 Trial

Background: To develop a new treatment modality, we conducted a phase 3 randomised trial of personalized peptide vaccination (PPV) for human leukocyte antigen (HLA) -A24-positive patients with castration-resistant prostate cancer (CRPC) for whom docetaxel chemotherapy failed. Methods: This randomized, double-blind, placebo-controlled, phase 3 trial was done at 68 medical centers in Japan. Eligible patents were aged 20 years or older with HLA-A24-positive, CRPC progressing after docetaxel chemotherapy, positive immunoglobulin G (IgG) responses to at least 2 of 12 warehouse peptides, an Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of at least 12 weeks, serum testosterone level of ≤ 50 ng/dl, and satisfactory bone marrow function, hepatic function, and renal function. Patients were randomly assigned in a 2:1 ratio to receive PPV or placebo. Four of 12 warehouse peptides selected based on pre-existing peptide-specific IgG levels or the corresponding placebo were subcutaneously injected in 6 doses weekly and then bi-weekly following the maximum of 30 doses until disease progression. The primary end point was overall survival (OS), and secondary end points were progression-free survival (PFS) and immune responses. Efficacy analyses were by the full analysis set. Findings: Between August 2013 and April 2016, 310 patients were randomly assigned (207 to PPV and 103 to placebo), and 306 patients were analyzed by the full analysis set (204 to PPV and 102 to placebo). Baseline characteristics were balanced between groups. The estimated median OS was 16.1 months (95% confidence interval [CI], 13–18·2) with PPV and 16·9 months (95% CI, 13·1–20·4) with placebo (hazard ratio [HR], 1·04; 95% CI, 0·80–1·37; p=0·77). The median PFS was not significantly different between groups. Grade < 3 adverse events were observed in 41% in both groups. The most common AEs were < grade 3 injection site reactions in both groups. The analysis of treatment arm effects among subgroups revealed lower HRs for OS in favor of the PPV arm in patients with < 64% neutrophils (HR, 0·55; 95% CI, 0·33–0·93, p=0·03) or ≥ 26% lymphocytes (HR, 0·70; 95% CI, 0·52–0·92, p=0·02) at base line by the significant interaction test (p=0·003 or p=0·007). Interpretation: PPV did not prolong OS or PFS in HLA-A24-positive patients with CRPC progressing after docetaxel chemotherapy. Subgroup analysis suggested that the patients with a lower proportion of neutrophils or a higher proportion of lymphocytes at base line can receive survival benefits from PPV treatment. Trial Regstration: This study is registered with UMIN Clinical Trials Registry (UMI11308), and patient enrollment is complete. Funding Statement: This study was supported by grants from the Japan Agency for Medical Research and Development (No. 18im0110802h0008) and Fujifilm Company. Declaration of Interests: MN has served as an advisory board consultant for BrightPath biotherapeutics Co. Ltd. KI received research funding from Taiho Pharmaceutical Company. SN has served as a consultant to BrightPath biotherapeutics Co. Ltd and received an honorarium from Sanofi. All other authors declare no competing interests. Ethics Approval Statement: The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The protocol was approved by institutional review boards or ethical committees at all of the institutions. All patients were Japanese and provided written informed consent before participating in this study.

Analysis and observation of T lymphocyte subsets and curative effects in patients with acquired pure red cell aplasia

Objectives To investigate distribution of T lymphocyte subsets, absolute value and proportion of large granular lymphocyte(LGL) in peripheral blood and bone marrow of acquired pure red cell aplasia (PRCA) patients, and to evaluate patients′ immune function and efficacy of immunosuppressive agents. Methods From January 2002 to November 2015, a total of 25 patients with acquired PRCA who were treated at Department of Hematology, People′s Hospital of Xinjiang Uygur Autonomous Region at first-visit were enrolled into this study, as study group. Inclusion criteria of study group: patients′ diagnoses were conformed to the diagnostic criteria of PRCA in Diagnosis and Curative Effect of Hematology (3rd edition) and clinical data were complete. Exclusion criteria: other anemia disease and clinical data were not complete. In 25 cases of PRCA patients, 5 cases were secondary PRCA with T cell-large granular lymphocyte leukemia (T-LGLL), other 20 cases were primary PRCA. And 25 cases of health people who were receiving physical examination at the People′s Hospital of Xinjiang Uygur Autonomous Region during the same period were recruited into control group randomly, by simple random method. Inclusion criteria of control group: no abnormality in blood routine examination and biochemical test. Exclusion criteria: abnormality in blood routine examination, and suffering from immune disease, tumor. T lymphocyte subsets of subjects in study group and control group were detected by flow cytometry. Blood routine examination and bone marrow examination were carried out in study group before and after treatment. Then absolute value and proportion of LGL in bone marrow, peripheral blood and curative effect after drug treatment of cyclosporine A and so on of 25 acquired PRCA patients were analyzed retrospectively and compared statistically. The study protocol was approved by the Ethical Review Board of Investigation in Human at People′s Hospital of Xinjiang Uygur Autonomous Region. There were no statistically significant differences of subjects′ clinical data, such as age and gender ratio, between two groups (P>0.05). Results ①Compared with control group, helper T lymphocyte (Th) proportion and Th/suppressor T lymphocyte (Ts) ratio of acquired PRCA patients in study group were significantly lower, Th proportion: (36.6±3.8)% vs (45.1±2.1)%; Th/Ts ratio: 1.2±0.2 vs 2.2±0.4; there were significant differences (t=9.161, 12.174, P=0.032, 0.021), and Ts proportion of study group was significantly higher than that of control group, (30.5±2.8)% vs (20.2±1.9)%; there was significant difference (t=13.460, P=0.021). There was no statistically significant difference of natural killer (NK) cell proportion between two groups, (11.3±1.8)% vs (10.3±1.3)%; and t=1.572, P=0.344. In study group, Th proportion, Th/Ts ratio of secondary PRCA patients with T-LGLL were lower than those of primary PRCA patients, Th proportion: (36.1±3.7)% vs (39.1±6.1)%; Th/Ts ratio: 1.0±0.2 vs 1.2±0.1; but the differences were not significant (t=2.293, 2.513; P=0.301, 0.297), and Ts proportion of secondary PRCA patients with T-LGLL was higher than that of primary PRCA patients, (31.0±2.7)% vs (28.4±2.0)%; but the difference was not significant (t=1.472, P=0.384). There was no statistically significant difference of NK cells proportion between two groups, (11.3±1.8)% vs (11.1±2.7)%; and t=1.572, P=0.364. ② Among 25 acquired PRCA patients in study group, 16 cases had higher proportion of lymphocytes in peripheral blood (64%, 16/25), and 10 cases had 2~3 granules LGL (40%, 10/25); 8 cases had more than 20% LGL of total lymphocytes (32%, 8/25). Four cases had 2~3 granules LGL in bone marrow (16%, 4/25); 3 cases had more than 20% LGL of total lymphocytes (12%, 3/25). The ratio of patients with LGL in peripheral blood was significantly higher than that in the bone marrow (χ2=6.595, P=0.003). ③ After treatment, hemogram of 18 cases returned to normal, including four cases with normal myelogram, fourteen cases with decreased bone marrow erythroid cell hyperplasia, which was higher than that before treatment. Effectiveness evaluation of study group was as follows: four PRCA patients were cured basically; fourteen cases achieved remission; six cases achieved significant progress; one case was invalid. The total effective rate of treatment was 96% (24/25). Five secondary PRCA patients with T-LGLL didn′t need blood transfusion after treatment, but unlike primary PRCA patients, their hemoglobin levels didn′t recover to normal reference range. Conclusions The disorder of T lymphocyte subsets in patients with acquired PRCA caused the disorder of cellular immune function, and LGL detected in peripheral blood would help the diagnosis of acquired PRCA. Effect of cyclosporin A treatment on acquired PRCA was very significant, but curative effect of secondary PRCA patients with T-LGLL was poorer than that of primary PRCA. Key words: Red-cell aplasia, pure; Anemia, aplastic; T-lymphocyte subsets; Leukemia, large granular lymphocytic; Cyclosporine

Factors affecting leucocyte profiles in the little auk, a small Arctic seabird

AbstractLeucocyte profiles, especially the heterophil/lymphocyte ratio (H/L), are widely used to assess immune function and stress in birds. In this study, we investigated whether leucocyte profiles in a small colonial seabird, the little auk (Alle alle), are related to age (chicks, adults), sex or biometric traits, including body mass. We blood-sampled chicks at 4–8 and 14–18 days of life and chick-feeding adults with 14–20- and 24–30-day-old chicks. Relative numbers of heterophils and H/L generally differed among chicks and adults, with the highest values in adults with 14–20-day-old chicks and the lowest ones in 4–8-day-old chicks. Females had a higher proportion of lymphocytes than males. The relative numbers of eosinophils were similar in all the groups. The percentage of monocytes tended to differ among the age categories. Analyses of chicks revealed that the percentage of heterophils and the H/L ratios were significantly higher in older than in younger chicks. The opposite pattern was recorded for lymphocytes. In 14–18-day-old chicks, H/L (stress level) was negatively related to body mass, head-bill length and bill width. This time period is energetically stressful as nestlings start to exercise their wings outside the nest chamber. This suggests that the origin of stress in nestlings was mainly nutritional. We found a significant positive correlation between body mass at fledging and the relative number of eosinophils at 14–18 days of life, indicating a higher level of stress in lighter nestlings. The significant relationship between fledging age and the percentage of monocytes at 14–18 days of life suggested a poorer state of health in chicks that fledged at an older age.

Sarcoidosis in a Patient with Systemic Sclerosis and Primary Biliary Cirrhosis

A 73-year-old woman who had been diagnosed with systemic sclerosis was admitted for further examination of bilateral hilar lymphadenopathy. Sarcoidosis was confirmed based on elevated serum levels of angiotensin-converting enzyme, a high proportion of lymphocytes and a high CD4/CD8 ratio in bronchoalveolar lavage fluid, abnormal (67)Gallium uptake in the mediastinum and noncaseating granulomas in skin biopsy specimens. In addition, high levels of antimitochondrial M2 antibodies and alkaline phosphatase indicated primary biliary cirrhosis (PBC). Here we describe a rare triplex of sarcoidosis, SSc and PBC. Although the etiology of this complex remains unknown, these three diseases might share some pathogenesis.

Adenoid as a source of lymphocytes in the surface secretions of nasopharynx

The significance and protective efficacy of surface secretions on mucosal membranes in the upper airways are well recognized. The aim of our study was to reveal the role of the adenoids as a source of cellular components in the mucosal secretion. The adenoid removed because of its hypertrophy and the samples of surface secretions taken by "imprint method" described by Ebenfelt et al. from the group of 38 children were examined by flow cytometry. The lymphocyte subsets with following antigens: CD3, CD4, CD8, CD19, CD23, CD16+56, CD45RA, CD45RO, HLA-DR were analyzed. Two groups were studied: (1) AH, children with adenoid hyperplasia without otitis media; (2) OME, children with adenoid hyperplasia and persistent otitis media with effusion. Age-related changes in percentages of lymphocyte subpopulations were analyzed. The percentage of lymphocyte subsets in adenoid and secretion were similar, however, in the surface secretion there was higher proportion of lymphocytes B and NK and lower of T. There was no correlation between percentages of lymphocyte in adenoid and surface secretion except for lymphocytes Th with memory phenotype and lymphocytes B with marker CD23(+). In adenoid the percentage of T and Th lymphocytes increased with age and the percentage of B lymphocytes decreased. In the secretion age-related changes were less pronounced and only the percentage of lymphocytes B CD23(+) showed positive correlation with age. In cases of otitis media with effusion there was higher proportion of lymphocytes B (CD19(+) and CD19(+)CD23(+)) and lower of lymphocytes Ts and Th with naive phenotype in the secretion. In adenoid however, the cases of OME revealed lower percentage of lymphocytes B and higher of lymphocytes Ts. Similar proportions of lymphocyte subpopulations in surface secretion and in adenoid imply that adenoid is the source of those cells. However, lack of correlation between the percentages of lymphocytes in secretion and in adenoid suggest that the process of lymphocyte migration has active and selective character.

Real-Time Identification of Leukocyte Subsets and Cell Surface Receptor Microdomains in the Microvasculature of Wild-Type and Sickle Cell Mice In Vivo.

Intravital microscopy has contributed enormously to the recent mechanistic advances in leukocyte (WBC) trafficking. However, current methods have provided little knowledge about differential WBC subset behavior or the spatial redistribution of surface adhesion molecules during recruitment and activation. Here, we use digital high-speed multichannel fluorescence intravital videomicroscopy to identify WBCs and localize cell surface molecules. Adherent WBCs were ascertained using a series of near-simultaneous 4-channel images in randomly selected venular segments following the injection of low-dose (0.02–0.12 mg/kg) fluorescently labeled Mab against CD45, Gr-1, and F4/80. Preliminary experiments on FAC-sorted WBCs revealed that Gr–1+F4/80- cells were highly enriched in neutrophils (PMNs, ~99%), whereas the F4/80+ fraction was mostly monocytes (~70%) and the fraction negative for both markers was largely lymphocytes (~88%). In the cremaster muscle of C57BL/6 mice (n=6), 39 ± 4% of adherent CD45+ cells recruited in venules ~90 min following surgery were Gr–1+F4/80- (PMNs), and 45 ± 7% were F4/80+ (monocytes), while 18 ± 3% were Gr–1-F4/80- (lymphocytes). In contrast, the proportion of PMNs was significantly increased in cremasteric venules of sickle cell (SS) mice (Berkeley) (79 ± 7%, n=5; p<0.001 compared to C57BL/6). Our previous studies have revealed that the interactions between erythrocytes (RBCs) and adherent WBCs correlate with vascular occlusion and mortality in SS mice (Turhan et al, PNAS, 2002). To determine which WBC subset can capture RBCs in vivo, we monitored RBC-WBC interactions in high-resolution brightfield movies acquired immediately after the capture of fluorescence images. We found that the vast majority of interactions (~93%) occurred with CD45+Gr–1+F4/80- PMNs. The high proportion of lymphocytes in systemic venules was unexpected. We thus analyzed directly lymphocyte subclasses with antibodies specific for B and T cell markers (B220 and CD4/CD8). The proportion of B and T cells was consistent with the above results (total ~20%), similar between C57BL/6 and sickle cell mice, with B cells predominating in both groups (70–76% of all lymphocytes). Since the bone marrow (BM) is a target of SS disease, we examined differential WBC recruitment in this vascular bed. In contrast to the cremasteric microcirculation, CD45+Gr–1-F4/80- lymphocytes represented the major WBC subset in both C57BL/6 and SS venules (49 ± 2% and 63 ± 5%, respectively.) Adherent CD45+B220+ B lymphocytes were significantly more numerous than CD45+CD4/CD8+ T lymphocytes in C57BL/6 (6-fold ↑, p<0.001) and SS (9-fold ↑, p<0.005) mice. We next evaluated the spatial distribution of LFA-1 and PSGL–1 on Gr–1+ WBCs recruited in TNF-α-stimulated cremasteric venules. We observed that PSGL–1 distribution was polarized in the great majority of adherent Gr–1+ cells (93 – 97%), whereas neither LFA–1 nor Gr–1 was polarized. The majority of PSGL–1 clusters was luminal rather than adjacent to the endothelium in both stationary and slowly migrating cells. In stationary cells, PSGL–1 appeared random in relation to the direction of blood flow. By contrast, in migrating cells, PSGL–1 redistribution was strongly associated with the direction of cell migration, with PSGL–1 clusters being almost exclusively located at the trailing edge. In summary, our results reveal a profound difference in the WBC subsets recruited in the cremastric and BM venules, and a powerful method to analyze the formation of discrete microdomains in vivo during cell activation.

Pitfalls in the diagnosis of childhood leukaemia.

Pitfalls in the diagnosis of childhood leukaemia.

Leukocyte Counts in Cerebrospinal Fluid with the Automated Hematology Analyzer CellDyn 3500 and the Urine Flow Cytometer UF-100

The counting of leukocytes and erythrocytes in cerebrospinal fluid (CSF) is still performed microscopically, e.g., using a chamber in most laboratories. This requires sufficient practical experience, is time-consuming, and may constitute a problem in emergency diagnostics. Specific automated systems for CSF cell counting are not available at present. We tested the hematology analyzer CellDyn 3500 (CD) and the urine flow cytometer UF-100 (UF), which are not designed for CSF analysis. We studied >104 samples with both analyzers, and the counts obtained were compared with the reference method (Fuchs-Rosenthal chamber). Good linearity in the medically relevant range of 15 x 10(6) to 1000 x 10(6) leukocytes/L and a high degree of within-run accuracy were seen for both analyzers. Cell counting on the UF was excellent, especially when low cell counts were encountered (CV, 4. 9% compared with 28% observed for the CD). Method comparison showed that identical results could be detected for a majority of the count pairs. For a few samples, there was a discrepancy between the results from the analyzers and the counting chamber. In most cases, these were CSF samples containing a high proportion of lymphocytes. For these samples, the CD result led to a false-positive high leukocyte count, and on the UF these cells were not allocated to the leukocyte population, thus leading to false-negative counts. Both analyzers should not be used for CSF cell counting in all cases at present. However, once the technical and software problems have been solved, routine use of the two analyzers for CSF analysis should be seriously contemplated.

Effect of age on plasma membrane asymmetry and membrane fluidity in human leukocytes and platelets.

We determined whether ageing changes in plasma membrane phospholipid asymmetry were related to changes in membrane physical characteristics. Plasma membrane asymmetry was determined in polymorphonuclear leukocytes (PMN), lymphocytes, and platelets from 45 healthy young (mean 29 years, 26 male) and 28 healthy elderly (mean 70 years, 15 male) subjects by flow cytometric measurement of annexin V binding to cell surface phosphatidylserine. Membrane fluidity in lymphocytes and platelets from young and elderly subjects was determined by fluorescence polarization of 1,6-diphenyl- 1,3,5-hexatriene (DPH) and (4-trimethylammonium)-DPH (TMA). In elderly subjects, a higher proportion of lymphocytes had specific annexin V binding to phosphatidylserine (PS) than in young subjects (young: median percentage of cells with specific annexin V binding to PS 5.3 [second to fourth quintiles range 3.8-8.7]; elderly: 8.5 [5.2-17.2]; p = .028). No ageing change in annexin V binding to PMN was observed (young: 35.0% [21.8-53.5]; elderly: 39.6% [27.4-69.8]; p = .42). Platelets had no specific annexin V binding (young: median molecules of annexin V specific binding 3.8 [0.4-11.3]; elderly: -1.4 [-4.8-1.7]; p = .23). Superficial membrane fluidity was increased in lymphocytes (TMA anisotropy, young: 0.271 [0.259-0.289]; elderly: 0.262 [0.242-0.279];p = .004), but not in platelets (young: 0.273 [0.259-0.293]; elderly: 0.269 [0.248-0.284]; p = .12). Lymphocyte annexin V binding correlated with TMA (r = -.65, p = .022), but not DPH anisotropy (r = -.39, p = .18). Plasma membrane asymmetry is decreased with age in human lymphocytes, but not in human PMN or platelets. The increased proportion of lymphocytes with loss of plasma membrane asymmetry corresponds to the ageing changes in superficial membrane fluidity observed in lymphocytes. Such alterations in lymphocyte plasma membrane structure with age could account for changes in membrane-bound receptor function described with ageing, and may contribute to alterations in immune responsiveness and vascular thrombosis seen in older humans.

Characterization of a canine CD44 specific monoclonal antibody.

Monoclonal antibodies (mAbs) were generated against a CD44 mRNA expressing (RT-PCR) macrophage/monocyte cell line (DH82) from a dog with malignant histiocytosis. The mAbs, that reacted with DH82 cells by FACS analysis were tested on formalin-fixed, paraffin-embedded tissues. Exclusively the incubation of DH82 cell pellets with mAbs from clone 2D10 resulted in a cell membrane associated immunoreaction. Immunoelectron microscopy specified, that the antibody bound exclusively to the cell membrane and processes of DH82 cells. The mAb was tested on a variety of normal canine tissues, including lymphoid, urinary, alimentary, respiratory, and endocrine organs, nervous tissues, liver, pancreas, skin, and muscles. Furthermore, tumour and inflamed tissues were tested for immunoreaction with the mAb. Immunohistologically, the 2D10 mAb reacted with macrophages/monocytes, subsets of lymphocytes, epithelial cells, and central nervous system white matter. FACS analysis of canine peripheral blood leukocytes showed, that a high proportion of lymphocytes and granulocytes were positive with this mAb. Western blot analysis revealed, that the 2D10 mAb bound to a protein with a molecular weight of about 85 kDa. The results of FACS and Western blot analyses, RT-PCR, immunohistology and immunoelectron microscopy strongly suggest that the antigen detected by the 2D10 mAb is most likely the canine equivalent of human CD44, a cell bound hyaluronan binding proteoglycan.

Elevated B cells in sputum of asthmatics. Close correlation with eosinophils.

Sputum examination is a useful noninvasive method to study airway inflammation. We investigated the reproducibility and validity of the measurements of lymphocyte subsets in the sputum of 11 stable patients with asthma and 10 nonasthmatic smokers. Sputum was dispersed with 0.1% dithiothreitol. A differential cell count was made with Wright's stain. Aliquots were stained with antibodies to CD19 (B cells), CD3 (T cells), CD4 (helper), CD8 (suppressor), and the activation marker CD25 (IL2 receptor) on T-cell subsets and were assayed by flow cytometry. Sputum from patients with asthma compared with nonasthmatic subjects had more eosinophils (mean +/- SEM, 32.5 +/- 8.5 versus 1.3 +/- 0.5%, p < 0.01) and a higher proportion of lymphocytes that were B cells (16.2 +/- 3.2 versus 4.0 +/- 1.0%, p < 0.01), and these correlated closely with the eosinophils (r = 0.8, p < 0.01). Patients with asthma also had more activated T-helper cells (39.3 +/- 4.6 versus 9.0 +/- 9.0%, p = 0.05), but the comparison was limited to two smokers because of macrophage autofluorescence. The repeatability of measurements of helper T-cells (R = 0.94), suppressor T cells (R = 0.88), and activated helper T cells (R = 0.77) was good; repeatability of measurements of T and B cells could not be examined because these were reciprocals of each other. Asthmatic sputum has different lymphocyte profiles than sputum from nonasthmatic smoking control subjects. The results demonstrate a potential importance of antibody-producing lymphocytes in the airway and their relation to sputum eosinophilia in asthma.

Pulmonary sarcoidosis in children: serial evaluation of bronchoalveolar lavage cells during corticosteroid treatment.

The clinical course of sarcoidosis in children has not been well defined. Eight children with symptomatic sarcoidosis included in this study underwent pulmonary function tests and bronchoscopy with bronchoalveolar lavage (BAL) before treatment and during steroid therapy. At the start of therapy, functional parameters, mostly dynamic lung compliance and lung transfer factor for CO, were impaired. This was associated with abnormalities of BAL cell populations: increased total cell number with a high proportion of lymphocytes, modifications of lymphocyte subpopulation with an elevated CD4+/CD8+ ratio, and enhanced ability of alveolar macrophages to release hydrogen peroxide. Although respiratory abnormalities seemed to be similar at the initial stage of sarcoidosis in children and adults, the course of the disease appeared to be different. Despite the absence of respiratory symptoms and disappearance of chest radiographic abnormalities on prolonged steroid treatment, we found slow improvement of pulmonary functions associated with persistence of BAL lymphocytosis and elevated CD4+/CD8+ ratios. However, the ability of alveolar macrophages to release hydrogen peroxide was significantly reduced after 6 months of steroid treatment, and it remained identical to the control group. Therefore, the evaluation of disease activity appears to be critical for therapy in pediatrics, and for this purpose studies of alveolar macrophage activation may be of particular interest.