Detection of histidine decarboxylase in peripheral blood leukocytes by flow cytometry

Inflammation Research -     

Detection of ganciclovir resistance after valacyclovir-prophylaxis in renal transplant recipients with active cytomegalovirus infection

Whether valaciclovir (VCV) prophylaxis could be responsible for ganciclovir (GCV)-resistance of Human cytomegalovirus (HCMV) in transplantation has never been documented. A multicentric retrospective pilot study was undertaken to detect GCV-resistance through mutations within the UL97 gene in renal transplant recipients who experienced active HCMV infection and received valacyclovir prophylaxis. Twenty-three patients who experienced HCMV antigenaemia or DNAemia during or at the end of prophylaxis were included. UL97 genotyping was carried out on peripheral blood samples, using a nested in-house PCR, which amplified the full-length UL97 gene. One patient has a resistance-related mutation (M460I); the major risk factor for emergence of resistance in this patient was the presence of early and persistent antigenaemia. GCV-resistance during VCV-prophylaxis was rare after renal transplantation. However, special attention must be paid to patients developing early active HCMV infection under prophylaxis.     

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI‐H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age‐matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP‐PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04–0.24) were significantly higher (p<0.01) relative to their age‐matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01–0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families. Hum Mutat 31:317–324, 2010. © 2010 Wiley‐Liss, Inc.     

Chemotaxis of peripheral blood leukocytes in patients with diffuse purulent peritonitis

Leukocytic chemotaxis was evaluated in 43 patients with diffuse purulent peritonitis. It was found out that time course changes in chemotactic activity of leukocytes have a phasic pattern: it was reduced in an early postoperative period, there is a tendency to enhancement of cellular spontaneous and target migration 3-6 days after operation and onwards, maximal response to chemotactic stimulus on day 7-14 after operative intervention. Postoperative serum factors show a diverse influence on leukocytic chemotaxis. Correlation observed between chemotaxic activity and postoperative developments allows the author to propose an assessment of leukocytic chemotaxis in patients with diffuse purulent peritonitis on follow-up to diagnose early complications and to control immunocorrective therapy administered.     

乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测

背景：肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论。 目的：检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达。 方法：采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达。 结果与结论：肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×10⁴,(4.158~6.234)×10⁴ 拷贝/mL。肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例。6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性。提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏。     

Imbalances in peripheral blood T-cell subpopulations in renal transplant patients

The distribution of T-lymphocyte subpopulations bearing receptors for the Fc portion of IgG (TG) or IgM (TM) was monitored in 22 renal allograft recipients treated with immunosuppressive therapy and in 10 uraemic patients on haemodialysis. No significant difference in the distribution of T cells and T-cell subsets was found between normal controls and haemodialysed patients. In transplanted patients, however, a significant reduction of the total T-cell percentage (P less than 0.005), of TM subset percentage (P less than 0.025) and absolute number (P less than 0.005) and of TG absolute number (P less than 0.05) was observed. Considering patients with allografts functioning for more than 1 year only, the reduction in TM cells in terms of percentage (P less than 0.0005) and absolute number (P less than 0.025) was significant, while TG subset levels did not change significantly. In patients transplanted less than 1 year previous to our study, total T cells and T-cell subsets were reduced significantly only as absolute numbers. During the 1st year we observed several increments of TM values towards normal levels, especially in the first 2 months after transplantation. During this period, TM subset levels sharply increased at acute rejection crisis and returned to previous values with rejection reversal. Our results suggest that the TM subset plays a prominent role in the mechanisms involved in the immunological response to allografts, and therefore repeated TM cell monitoring could be useful in the follow-up of renal transplant patients.     

免疫相关基因在绝经后骨质疏松症患者外周血白细胞中的表达

文题释义： RNA-seq技术：利用高通量测序技术进行转录组测序分析,检测mRNA、miRNA、circRNA及lncRNA等所有转录本的表达水平,从而探究特定组织或细胞中基因的结构和功能,探寻特定疾病发生、发展的分子机制和信号通路。 骨免疫学：2000年首次提出,认为骨骼是一种免疫器官,在骨髓中形成的多种免疫细胞可与骨骼系统的细胞相互作用,通过经典的RANKL/RANL/OPG系统等信号通路共同调控骨代谢过程。最近提出的“免疫疏松”,进一步揭示骨免疫学在骨质疏松症中的调节作用。   摘要 背景：目前认为骨骼是一种免疫器官,在骨髓中形成的多种免疫细胞可与骨骼系统的细胞相互作用,共同调控骨代谢。研究绝经后骨质疏松症的发病机制,寻找其治疗的分子靶标和信号通路,有助于其预防和治疗。 目的：利用RNA-Seq技术,探讨绝经后骨质疏松症患者外周血白细胞中与免疫相关的基因表达谱的变化。 方法：选取因发生骨折住院治疗的绝经后0-20年的女性40人,首先采用Lunar Prodigy双能X射线骨密度仪扫描后,分为骨量正常组(T>-1)和骨质疏松组(T<-2.5)。全转录组测序(RNA-seq)分别抽取2组各5人的外周血样本,分离白细胞后提取总RNA,筛选2组的表达差异基因并进行GO富集分析,筛选免疫相关表达基因并进行KEGG信号通路分析,收集2组各20人的外周血样本采用Real-time PCR验证KEGG PATHWAY生物信息学分析的结果。研究方案的实施符合上海市浦东新区公利医院的相关伦理要求(20170301)。 结果与结论：①骨质疏松组与骨量正常组相比,表达差异在2倍以上且具有统计学意义的基因为187个(其中表达上调131个,表达下调56个),其中与免疫相关的表达差异基因为29个(其中表达上调25个,表达下调4个);②KIR3DL1、KIR3DL2、KIR2DL4、KLRD1及HSPA6在2组中的表达差异有显著性意义(P < 0.05);③结果说明,KEGG信号通路分析显示上述5个基因在自然杀伤细胞介导的细胞毒作用中发挥重要作用。KIR3DL1、KIR3DL2、KIR2DL4、KLRD1及HSPA6参与的自然杀伤细胞介导的细胞毒作用可能与绝经后骨质疏松症的发生密切相关。ORCID: 0000-0003-4879-0905(陈天宁) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

Interferon synthesis by peripheral blood leukocytes of patients with bronchial asthma

Interferon production by leukocytes of 28 bronchial asthma patients and 27 normal subjects was examined using whole blood technique. Interferon production in blood samples was induced by classical inducers and the obtained interferons were tested in A549 cells using EMC virus as challenge. Leukocytes from both atopic and infectious asthma patients showed decreased ability to interferon production in comparison to healthy donors. In atopic asthma statistically significant differences in interferon production induced by NDV, PHA + PMA and LPS were observed. In the case of infectious asthma lower amounts of interferon were noted after stimulation with LPS and PHA + PMA. A decrease in spontaneous interferon production was also observed.     

Analysis of the thiol status of peripheral blood leukocytes in rheumatoid arthritis patients

Although the exact etiology of rheumatoid arthritis (RA) remains unknown, there is increasing evidence that reactive oxygen species and a pro-oxidant/antioxidant imbalance are an important part of the pathogenesis of joint tissue injury. Flow cytometry was used to evaluate the thiol status [surface-thiols and intracellular glutathione (iGSH)] of leukocytes from RA patients and controls. Levels of surface-thiols and iGSH of leukocytes from RA patients were significantly lower than of leukocytes from controls. CD53, a glycoprotein of the tetraspanin superfamily, which coprecipitates with the GSH recycling enzyme gamma-glutamyl transpeptidase, was elevated significantly on leukocytes from RA patients compared with leukocytes from controls. Surface-thiols and GSH play important roles in redox buffering of cells, providing protection from oxidative stress. The chronic inflammation of RA has been associated with oxidative stress, which is shown to cause a decline in the levels of cellular antioxidant sulfhydryls (R-SH). As antioxidant-protective levels also decline with age, the problem is compounded in older RA patients, who did have fewer R-SH. Chronic stress can also have an effect on telomere lengths, determining cell senescence and longevity. Although telomeres shorten with increasing age, our flow cytometry studies indicate that accelerated shortening in telomere lengths occurs with increasing age of RA patients, suggesting premature cellular aging. The paradox is that lymphocytes from RA patients are believed to resist apoptosis, and we suggest that the elevated expression of CD53, which results from the increased oxidative stress, may protect against apoptosis.     

Evaluation of apoptotic leukocytes in peripheral blood smears

OBJECTIVES: Apoptosis is a phenomenon of physiological cell death in which each step is regulated, similar to the process of mitosis. We observed apoptosis in leukocytes during the review of peripheral blood smears. This study was undertaken to evaluate the morphologic features of apoptotic leukocytes in peripheral blood smears and to ascertain their clinical significance. DESIGN: Sixty cases (23 males and 37 females, aged newborn to 92 years) exhibiting apoptotic leukocytes in peripheral blood smears were studied. Medical records for each case were reviewed, and patients were categorized according to their clinical diagnoses. RESULTS: Neutrophils were the most common apoptotic leukocytes identified (85%), followed by lymphocytes (18%) and eosinophils (2%). The diagnosis most frequently associated with the presence of apoptotic leukocytes was infection (55%). Apoptosis in lymphocytes was comparatively less common, but when present, the most common associations were diabetes mellitus, glucocorticoid administration, and neoplastic diseases. CONCLUSIONS: Our findings suggest that the presence of apoptotic leukocytes in peripheral blood smears may help in the differential diagnosis and may be related to the severity of disease. We recommend further evaluation using additional special techniques for detecting apoptotic leukocytes.     

Immunophenotype characteristics of peripheral blood mononuclear leukocytes of chronic idiopathic urticaria patients

The pathogenesis of chronic idiopathic urticaria (CIU) is not completely understood although autoimmunity has been proposed. The aim of the study was to assess the expression of different leukocyte antigens, by flow cytometry, assaying total blood of 29 patients with CIU and of 20 sex and age matched controls. Moreover, we assessed soluble CD154 a marker of immune cell activation, predominantly memory T cells. When patients were divided depending an their response to the autologous serum skin test (ASST), three different groups were encountered: group 1 (n=11): with negative ASST-, group 2 (n=11): positive ASST (ASST+) with normal lymphocyte counts and group 3 (n=7): ASST+ with low lymphocyte counts (< 1500 cells/mm3). A significant increase in CD19+ percentage and not in the absolute count (P < 0.05) was observed in group 1 as compared to controls and to the other groups. In contrast, CD30+, CD45RO+ and CD4+/CD45RO+ percentages and biologically active soluble CD154 levels were significantly higher (P < 0.05) in group 3 as compared to group 1 or to controls. In ASST positive groups, CD45RO+ and CD4+/CD45RO+ positiveness correlates with wheal diameter. In conclusion, memory cells may play a role in these different types of patients and in understanding CIU pathogenesis.     

Neutral endopeptidase activity in human peripheral blood leukocytes

Neutral endopeptidase (NEP; EC 3.4.24.11) efficiently hydrolyses many neuropeptides. To determine the distribution of NEP, a possible regulatory enzyme for the neuropeptide-induced leukocyte activation, among human leukocytes, we investigated the enzymatic activity of NEP in each cell type of human peripheral blood leukocytes. The activity of NEP assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils (59.0 +/- 9.1 pmol/min 10(6) cells); however, the NEP activity was virtually absent in mononuclear cells, eosinophils and basophils. Common acute lymphoblastic leukemia antigen (CALLA) detected immunocytochemically with three anti-CALLA antibodies, whose amino acid sequence has been shown to be identical with that of NEP, was also found only in neutrophils, but not in other blood leukocytes. It is suggested that NEP might regulate the neuropeptide-induced activation of human neutrophils.     

Pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir in allogeneic stem cell transplant patients with graft-versus-host disease of the gastrointestinal tract.

The pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir were compared in allogeneic stem cell transplant recipients with stable graft-versus-host disease of the gastrointestinal tract. Twenty-two evaluable adult patients were randomized to receive a single dose of open-label study drug (900 mg of oral valganciclovir or 5 mg/kg of intravenous ganciclovir). After a washout period of 2 to 7 days, patients were crossed over to receive the alternate study drug. Ganciclovir and valganciclovir concentrations in plasma were measured over 24 hours after dosing. Noninferiority of 900 mg of valganciclovir relative to intravenous ganciclovir was concluded if the lower limit of the 1-sided 95% confidence interval of the ratio of least-square means of the ganciclovir area under the curve (AUC) for the 2 study drugs was >80%. Valganciclovir was found to be rapidly absorbed and converted into ganciclovir. The ganciclovir exposure after 900 mg of valganciclovir noninferior to that of intravenous ganciclovir (AUC0-infinity, 52.1 and 53.8 microg.h/mL, respectively; 95% confidence interval of the ratio of least square means of AUC0-infinity, 82.48%-118.02%). Oral valganciclovir could be a useful alternative to intravenous ganciclovir in certain stable stem cell transplant patients who require prophylaxis or preemptive therapy for cytomegalovirus infection.     

Clinical correlations of peripheral blood microchimerism after liver transplantation

The aim of this study was to evaluate microchimerism after human liver transplantation (LT). This study included 13 female recipients who received hepatic allograft from male donors at Asan Medical Center. A nested PCR specific for Y-chromosome gene (DYZ3) was used to analyze the small number of male cells in the peripheral blood mononuclear cells of the female recipients. Microchimerism was observed in 6 of 13 recipients and 16 out of 35 samples. Only 3 patients showed microchimerism 3 months after LT. There was no statistical difference between the presence of microchimerism and clinical findings such as type of donor, type of immunosuppression, episode of rejection and age of recipient. This study did not show any clinical relevance of microchimerism and further larger study are needed to confirm the results.     

Systemic suppression of human peripheral blood phagocytic leukocytes after whole-body UVB irradiation.

We examined systemic effects of whole-body UVB irradiation on human peripheral blood phagocytes. We found that 24 h after a single erythemal dose of UVB radiation two phagocyte functions, adhesion and phagocytosis, were reduced by 50%. This functional suppression was accompanied by a significant decrease in the expression of complement receptors (CR1 and CR3) and IgG Fc receptors (FcRII and FcRIII). The greatest reduction (47%) was observed in CR3, which is important for both adhesion and phagocytosis. A kinetic analysis showed that both CR1 and CR3 levels started to decrease 15 min after the UVB exposure, reaching the lowest levels at 4.5- and 24-h time points, respectively. The down-modulation of CRs after whole-body UVB exposure was not due to a defective receptor synthesis or translocation from internal stores to plasma membrane because the maximal CR levels in stimulated cells were not affected by UVB. No change in the serum soluble ICAM-1 was detected after UVB, which rules out CD1 1b epitope masking by sICAM-1. UVB did not release low-receptor-density myeloid progenitor cells from storage pools into circulation. Interleukin 10, a mediator of UVB-induced immunosuppression, was unable to modulate CR expression in vitro. When seven suberythemal whole-body UVB exposures were given repeatedly within 2 weeks, a significant decrease in CR expression was seen, which was greatest after three irradiations. Our data suggest that an exposure to UVB has systemic effects in humans which, possibly due to the down-modulation of preexisting cell-surface receptors, suppress some important functions of circulating phagocytic cells.