Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.@*METHODS@#Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.@*RESULTS@#After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).@*CONCLUSION@#The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Clinical study of 19 cases of steroid-refractory gastrointestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with fecal microbiota transplantation / 中华血液学杂志

Objective: To investigate the clinical efficacy of fecal microbiota transplantation (FMT) for treating steroid-refractory gastrointestinal acute graft-versus-host disease (GI-aGVHD) . Methods: This analysis included 29 patients with hematology who developed steroid-refractory GI-aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Huaian Hospital Affiliated to Xuzhou Medical University from March 2017 to March 2022. Among them, 19 patients underwent FMT treatment (the FMT group) and 10 patients did not (the control group). The efficacy and safety of FMT were assessed, as well as the changes in intestinal microbiota abundance, lymphocyte subpopulation ratio, peripheral blood inflammatory cytokines, and GVHD biomarkers before and after FMT treatment. Results: ① Complete remission of clinical symptoms after FMT was achieved by 13 (68.4%) patients and 2 (20.0%) controls, with a statistically significant difference (P<0.05). Intestinal microbiota diversity increased and gradually recovered to normal levels after FMT and FMT-related infections did not occur. ②The proportion of CD3(+) and CD8(+) cells in the FMT group after treatment decreased compared with the control group, and the ratio of CD4(+), regulatory T cells (Treg), and CD4(+)/CD8(+) cells increased (all P< 0.05). The interleukin (IL) -6 concentration in the FMT group was lower than that in the control group [4.15 (1.91-5.71) ng/L vs 6.82 (2.40-8.91) ng/L, P=0.040], and the IL-10 concentration in the FMT group was higher than that in the control group [12.11 (5.69-20.36) ng/L vs 7.51 (4.10-9.58) ng/L, P=0.024]. Islet-derived protein 3α (REG3α) was significantly increased in patients with GI-aGVHD, and the REG3α level in the FMT group was lower than that in the control group after treatment [30.70 (10.50-105.00) μg/L vs 74.35 (33.50-139.50) μg/L, P=0.021]. Conclusion: FMT is a safe and effective method for the treatment of steroid-refractory GI-aGVHD by restoring intestinal microbiota diversity, regulating inflammatory cytokines, and upregulating Treg cells.

Progress in differentiation regulation and subpopulation typing of exhausted CD8 + T cells / 中华微生物学和免疫学杂志

Exhausted CD8 + T cells (CD8 + Tex) are a distinct subpopulation formed from naive CD8 + T cells under conditions of sustained high antigen stimulation. Initially, naive CD8 + T cells can differentiate into functional cytotoxic cells and exert anti-infective and anti-tumor effects upon short-term antigen stimulation. However, sustained high antigen stimulation will make effector CD8 + T cells progressively differentiate into terminally CD8 + Tex cells and irreversibly lose effector function. Unlike memory and effector T cells, CD8 + Tex cells have a unique transcriptional program. Numerous studies are attempting to map a detailed differentiation landscape of CD8 + Tex cell subsets, aiming to maximize the number of effector T cells in the future by targeting individual subsets or individual differentiation stages in CD8 + Tex cells without damaging the effector cells. This article reviewed the progress in CD8 + Tex cells from the aspects of transcriptional dysregulation, metabolic reprogramming, subpopulation typing and clinical application, aiming to provide more CD8 + T cell-based therapeutic strategies for tumor.

Immunoregulation of allograft rejection: A role played by human CD200+ sub-population from human placenta-derived mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Immune rejection of skin allograft is a clinical problem to be solved. Our previous study has shown that human placenta-derived CD200+ mesenchymal stem cells may have a strong capability of immunoregulation. OBJECTIVE: To investigate the effects of human placenta-derived CD200+ mesenchymal stem cells on rejection of skin allograft. METHODS: Skin allograft models of c57/BL6 mice were established and divided into three groups as control group, human placenta-derived mesenchymal stem cells group (PMSCs group), and CD200+ mesenchymal stem cells group (CD200+-PMSCs group). PBS (control group), normal PMSCs and CD200+-PMSCs were injected into the mice through the tail vein, respectively. The necrotic time, survival time and situation of grafted skin were observed. The number of peripheral white blood cells was counted after 7 days of treatment. The expression levels of interleukin-10, interferon-γ and tumor necrosis factor-α were detected by Q-PCR and ELISA. RESULTS AND CONCLUSION: (1) Compared with the control group, in the PMSCs and CD200+-PMSCs groups, the condition of skin allograft was better, and the survival time was significantly prolonged (P < 0.001). The condition and survival time of skin allograft in the CD200+-PMSCs group were significantly superior to those in the PMSCs group (P < 0.01). (2) After 7 days of treatment, the number of peripheral white blood cells in the PMSCs and CD200+-PMSCs groups was significantly less than that in the control group (P < 0.01). (3) Compared with the control group, the mRNA expression level of interleukin-10 in the spleen was significantly increased in the CD200+-PMSCs group (P < 0.05), and the mRNA expression levels of interferon-γ and tumor necrosis factor-α in the spleen were significantly down-regulated in the PMSCs and CD200+-PMSCs groups (P < 0.05, P < 0.01). The mRNA expression levels of interferon-γ and tumor necrosis factor-α in the spleen in the CD200+-PMSCs group were significantly lower than those in the PMSCs group (P < 0.01, P < 0.05). (4) Compared with the control group, the expression level of interleukin-10 in the blood was significantly increased (P < 0.05, P < 0.001), and the expression levels of interferon-γ and tumor necrosis factor-α in the blood were significantly down-regulated in the CD200+-PMSCs and PMSCs groups (P < 0.05, P < 0.001; P < 0.01, P < 0.001). The expression levels of interferon-γ and tumor necrosis factor-α in the blood in the CD200+-PMSCs group were significantly lower than those in the PMSCs group (P < 0.05). These results indicate that human placenta-derived mesenchymal stem cells have immunosuppressive effect on the rejection of skin allograft, and CD200+ cells may have better immunoregulatory effects by regulating the expressions of interleukin-10, interferon-γ and tumor necrosis factor-α

Application potential of CD146 positive subpopulation as seed cells for cartilage tissue engineering / 中国组织工程研究

BACKGROUND: The development of regenerative medicine and the appearance of tissue engineering technology provide a new solution for cartilage defect reconstruction. In tissue engineering, mesenchymal stem cells are widely used seed cells. However, as a heterogeneous cell group, stem cells play different roles in different subsets. Therefore, the application of key functional subsets of mesenchymal stem cells in cartilage repair has a broad application prospect. OBJECTIVE: To sort CD146 positive subpopulation cells from human adipose-derived mesenchymal stem cells to verify their biological characteristics and potential as seed cells in cartilage tissue engineering. METHODS: Human adipose-derived mesenchymal stem cells were provided by Zhejiang Jinshidai Biotechnology Co., Ltd. Surface markers of human adipose-derived mesenchymal stem cells were identified by flow cytometry. CD146 positive subpopulation cells were sorted from human adipose-derived mesenchymal stem cells using magnetic-activated cell sorting. Molecular characteristics of two kinds of cells were analyzed by gene chip detection technology and bioinformatics analysis technology. Two kinds of cells were induced to chondrocytes in vitro and histologically examined. Cell viability and apoptosis of two kinds of cells were detected before and after cryopreservation. RESULTS AND CONCLUSION: Human adipose-derived mesenchymal stem cells highly expressed stem cell-associated markers CD73 and CD90, but did not express hematopoietic stem cell-associated markers CD34, CD45, and HLA-DR. Bioinformatics analysis results showed that CD146 positive subpopulation had different functions in inflammatory pathways and musculoskeletal diseases compared with human adipose-derived mesenchymal stem cells. CD146 positive subpopulation could differentiate into cartilage, and its chondrogenic differentiation ability was better than that of human adipose-derived mesenchymal stem cells. CD146 positive subpopulation had better apoptosis and activity than human adipose-derived mesenchymal stem cells after resuscitation. These results suggest that CD146 positive subpopulation has good chondrogenic differentiation potential and is a promising seed cell for cartilage tissue engineering.

Aquios CL flow cytometer performance in the automated quantification of lymphocyte subpopulations / Desempenho do citômetro de fluxo Aquios CL na quantificação automatizada de subpopulações linfocitárias

ABSTRACT Flow cytometry (FC) is an essential tool for diagnosis, prognosis and therapeutic follow-up of several hematologic malignancies. In addition, it performs the quantification of lymphocytes subpopulations for diagnosis and monitoring of primary and acquired immunodeficiencies through the antigenic expressions of CD19 and CD20 for B lymphocytes; CD2, CD3, CD4, CD8 for T lymphocytes; and CD56 and CD16 for the identification of natural killer (NK) cells. The cytometry technique has revolutionized the way that the cells are identified, and over the years this platform has progressed with several advances in hardware and software that aim to improve workflow resulting in higher productivity, quality and cost savings. The Aquios CL - Beckman Coulter (BC) is an example of this advance because it is a complete automation instrument in flow cytometry called "Load & Go flow cytometer" for quantification of lymphocyte subpopulations in the routine diagnosis. In this study, the Aquios CL was validated, and quantification in frequency and absolute numbers of the lymphocyte subpopulations had an excellent correlation with the results obtained by the dual platform quantification performed in the Cytomics FC500 (BC) and automated Sysmex XE-2100 cell analyzer.

RESUMEN La citometría de flujo (CF) es una herramienta importante para diagnóstico, pronóstico y seguimiento terapéutico de diversas neoplasias hematológicas. Además, posibilita la cuantificación de las subpoblaciones linfocitarias (SPL) para asistencia diagnóstica y monitoreo de las inmunodeficiencias primarias y adquiridas a través de las expresiones antigénicas de CD19 y CD20 para linfocitos B; CD2, CD3, CD4 y CD8 para linfocitos T; y CD56 y CD16 para identificación de células natural killer (NK). La CF ha revolucionado la manera como las células son identificadas y, a lo largo de los años, esa plataforma ha progresado con diversos avances en hardware y software que aspiran a mejorar el flujo de trabajo resultando en mayor productividad, calidad y reducción de costos. El Aquios CL Beckman Coulter (BC) es un ejemplo de ese avance, pues es un instrumento de automación completa en CF (llamado Load & Go flow cytometer) para cuantificación de SPL en la rutina diagnóstica. En este estudio el Aquios CL fue validado, y la cuantificación en números relativos y absolutos de las subpoblaciones linfocitarias tuvo una excelente correlación con los resultados obtenidos por la cuantificación en plataforma dupla realizada en el Cytomics FC500 (BC) y en el analizador automatizado de células Sysmex XE-2100.

RESUMO A citometria de fluxo (CF) é uma ferramenta importante para diagnóstico, prognóstico e acompanhamento terapêutico de diversas neoplasias hematológicas. Além disso, possibilita a quantificação das subpopulações linfocitárias (SPL) para assistência diagnóstica e monitoramento das imunodeficiências primárias e adquiridas por meio das expressões antigênicas de CD19 e CD20 para linfócitos B; CD2, CD3, CD4 e CD8 para linfócitos T; e CD56 e CD16 para identificação de células natural killer (NK). A técnica de CF revolucionou a maneira como as células são identificadas e, ao longo dos anos, essa plataforma tem progredido com diversos avanços em hardware e software que visam melhorar o fluxo de trabalho, resultando em maior produtividade, qualidade e redução de custos. O Aquios CL - Beckman Coulter (BC) é um exemplo desse avanço, pois é um equipamento de automação completa em CF (denominado Load & Go flow cytometer) para quantificação de SPL na rotina diagnóstica. Neste estudo, o Aquios CL foi validado, e a quantificação em números relativos e absolutos das subpopulações linfocitárias teve uma excelente correlação com os resultados obtidos pela quantificação em plataforma dupla realizada no Cytomics FC500 (BC) e no analisador automatizado de células Sysmex XE-2100.

Determinación y cuantificación de subpoblaciones de linfocitos T y células natural killer en sangre periférica de individuos sanos por citometría de flujo / Detection and quantification of T-cell subpopulations and NK cells in peripheral blood from healthy individuals

Resumen Introducción: La determinación de las diferentes subpoblaciones de los linfocitos T en las diversas patologías y el monitoreo postratamiento ayuda a que el médico tome decisiones terapéuticas teniendo como referencia la cinética de los linfocitos T localizados en sangre periférica. Métodos: Se realizó la estandarización de un perfil de moléculas de superficie para la caracterización de subpoblaciones de linfocitos T: naïve, activados y de memoria, así como las células natural killer o asesinas naturales (CD3− CD56+) en sangre periférica de individuos clínicamente sanos. Resultados: Se identificaron las subpoblaciones de linfocitos: naïve (CD3+, CD4+ o CD8+, CD45RA+, CD62L+, CCR7+), activados (CD3+, CD4+ o CD8+, CD45RA+ o CD45RO+, CD69+ y/o CRTAM+), efectores (CD3+, CD4+ o CD8+, CD45RA+, CD62L−, CCR7−), de memoria central (CD3+, CD4+ o CD8+, CD45RO+, CD62L+, CCR7+) y de memoria efectora (CD3+, CD4+ o CD8+, CD45RO+, CD62L−, CCR7−) en las poblaciones de linfocitos T CD4+ y CD8+. Se integraron los datos obtenidos con estadística descriptiva (valores mínimos, valores máximos, media, mediana). Conclusiones: Este panel será de gran utilidad para monitorear pacientes en quienes se requiera valorar el estado inmunológico desde el punto de vista celular. Particularmente, puede apoyar en el seguimiento de los pacientes en los que se requiera evaluar la reconstitución inmunológica (componente celular de estirpe T).

Abstract Background: The knowledge of the participation of different subpopulations of T lymphocytes in various pathologies helps to make therapeutic decisions, having as reference the presence of the different subpopulations of the T lymphocytes associated with the disease. Methods: A profile standardization of surface molecules for the characterization of subpopulations of T cells was conducted: naïve, activated and memory, as well as natural killer (CD3− CD56+) cells in peripheral blood of clinically healthy individuals. Results: Naïve (CD3+, CD4+ or CD8+, CD45RA+, CD62L+, CCR7+), activated (CD3+, CD4+ or CD8+, CD45RA+ or CD45RO+, CD69+ and/or CRTAM+), effectors (CD3+, CD4+ o CD8+, CD45RA+, CD62L−, CCR7−), central memory (CD3+, CD4+ o CD8+, CD45RO+, CD62L+, CCR7+), memory effectors (CD3+, CD4+ or CD8+, CD62RO+, CD62L−, CCR7−) subpopulations were analyzed by flow cytometry. Descriptive statistics parameters were calculated (minimum values, maximum values, mean values, median). Conclusions: This panel can be very useful for monitoring patients in whom the immunological status from a cellular perspective is needed. Particularly, it can support the follow-up of patients who require an immunological reconstitution (T-cell component) evaluation.

Diagnostic value of flow cytometry in postoperative infection after renal transplantation / 器官移植

Objective To assess the value of flow cytometry in the diagnosis of postoperative infection following renal transplantation. Methods According to postoperative imaging findings and laboratory examination outcomes, 51 recipients undergoing the first renal transplantation were divided into the bacteria (n=33), fungus (n=9) and BK virus (n=9) groups. Twenty-eight recipients with stable conditions after renal transplantation were assigned into the stable group. Flow cytometry was adopted to detect the percentage and absolute counting of lymphocyte subpopulation in the peripheral blood of recipients in each group. Renal function, percentage and absolute counting of lymphocyte subpopulation in the peripheral blood were statistically compared among different groups. Receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of the percentage and absolute counting of lymphocyte subpopulation in infectious diseases after renal transplantation. Results Compared with the stable group, the serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the bacteria, fungus and BK virus groups were significantly up-regulated, respectively (P=0.035, 0.007, 0.024; 0.037, 0.006, 0.032). Compared with the stable group, the percentage of CD16+CD56+natural killer (NK) cells was significantly declined in the bacterial (P=0.036) and fungus groups (P=0.015), and the proportion of CD4+/CD8+T cells was dramatically decreased in the fungus group (P=0.004). Compared with the bacterial group, the percentage of CD3+CD8+T cells was significantly elevated (P=0.013 and 0.008), the proportion of CD3+CD4+T cells was considerably declined (P=0.003 and 0.010), and the percentage of CD4+/CD8+T cells was significantly declined (P=0.003 and 0.005) in the fungus and BK virus groups. Compared with the stable group, the quantity of CD3+T cells, CD3+CD8+T cells and CD16+CD56+NK cells was significantly declined in the bacterial, fungus and BK virus groups, respectively (P=0.025, 0.002, 0.003; 0.015, 0.005, 0.006; 0.001, 0.001, 0.031). In addition, the quantity of CD3+CD4+T cells was considerably decreased in the fungus and BK virus groups (P=0.001, 0.003). The quantity of CD19+B cells was significantly reduced in the BK virus group (P=0.019). Compared with the bacterial group, the quantity of CD3+CD4+T cells was considerably lower in the fungus group (P=0.023). ROC curve analysis revealed that the quantity of CD3+CD4+T cells [area under curve(AUC)=0.8492] and CD16+CD56+NK cells (AUC=0.8889) yielded relatively high accuracy in the diagnosis of fungal infection. The quantity of CD3+T cells (AUC=0.8472), CD3+CD4+T cells (AUC=0.8452) and CD19+B cells (AUC=0.8115) yielded relatively high accuracy in the diagnosis of BK virus infection. Conclusions Flow cytometry detection of the lymphocyte subpopulation in peripheral blood can evaluate the immune function of patients. Absolute counting of lymphocyte subpopulation can directly assess the degree of immunity. These two combined parameters provide guiding significance for the diagnosis and differential diagnosis of infectious diseases in recipients after renal transplantation.

Expression of granulocyte colony-stimulating factor receptor in colitis-associated colonic carcinogenesis / 中华肿瘤杂志

Objective@#To investigate the expression of granulocyte-colony stimulating factor receptor (G-CSFR) in a mouse model of colitis-associated cancer (CAC), and the roles of G-CSFR positive immune cells in the development of CAC.@*Methods@#The C57BL/6 mouse model of CAC was established by azoxymethane and dextran sulphate sodium. Three different stages in the development of CAC, including inflammation (AD1), mild dysplasia (AD2) and adenocarcinoma (AD3) were simulated. Colon tissue was digested into single cell suspension and the expressions of G-CSF and G-CSFR were analyzed by real-time PCR and fluorescence activated cell sorter (FACS). The expressions of G-CSFR on T cell, macrophage and neutrophil were analyzed by FACS.@*Results@#The establishment of mouse model can effectively simulate the disease progression of CAC. The results of real-time PCR detection showed that the expression level of G-CSF mRNA in AD1, AD2 and AD3 groups were 1.2, 7.3 and 18.0-fold changes of the control group, respectively. The differences between AD2, AD3 and control groups were statistically significant (P<0.05). G-CSFR mRNA levels in AD1, AD2 and AD3 groups were 1.5, 2.2 and 4.5-fold changes of the control group, respectively. The difference between AD3 and control groups was statistically significant (P<0.05). FACS showed that the percentages of CD45+ G-CSFR+ cells in colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.84±1.77)%, (41.48±4.15)%, (44.84±8.54)% and (57.76±1.95)%, respectively.The percentages of CD45+ G-CSFR+ cells in AD2 and AD3 groups were significantly higher than that of control group (P<0.05). The percentages of CD45+ G-CSFR+ macrophage in the colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.54±5.88)%, (47.14±5.25)%, (42.49±7.80)% and (29.25±8.24)%, respectively. The percentages of CD45+ G-CSFR+ T cells in these groups were (30.04±6.87)%, (29.65±8.08)%, (33.75±7.37)% and (33.32±9.85)%, respectively. The percentages of CD45+ G-CSFR+ granulocyte were (2.39±2.10)%, (4.05±1.56)%, (3.62±2.67)% and (2.26±0.85)%, respectively (P<0.05). The percentages of G-CSFR+ macrophage and G-CSFR+ T cells were significantly higher than that of G-CSFR+ granulocyte (P<0.05). The differences between AD1 and control group, AD2 and control group, AD1 and AD2 group, AD2 and AD3 group were statistically significant (P<0.05).@*Conclusions@#The expression of G-CSFR is significantly up-regulated in the development of CAC. The enrichment of G-CSFR+ macrophages in the colon tissue suggests G-CSFR+ macrophages participate in the development of CAC.

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus) / 亚洲男科学杂志(英文版)

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations - SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans.

Effects of Glycyrrhizic Acid Ointment on Th1/Th2 Immunologic Mechanism in Mice with Chronic Eczema / 医药导报

Objective To observe the immunological mechanism of glycyrrhizic acid ointment on chronic eczema mouse model. Methods Mice were randomly divided into blank control group,model control group,base material group, dexamethasone acetate cream group,glycyrrhizic acid ointment high,medium and low dose groups.Except for the blank control group,chronic eczema mouse model was established by chronic repeated 2,4-nitrochlorobenzene stimulation.The contents of IL-4, IL-18,IFN-γ in serum and the T lymphocyte subpopulation in the peripheral blood were all determined after 14 days. Results Glycyrrhizic acid ointment in high,medium groups could significantly reduce the contents of interleukin (IL)-2,IL-5,IL-6,IL-18,interferon(IFN)-γ in serum,with significant differences as compared with the model control group(P<0.01 or P<0.05).The content of IL-4 in serum was significantly increased in the glycyrrhizic acid ointment high,medium groups as compared with the model control group (P<0.01 or P<0.05).Compared with model control group,CD+4T lymphocyte percentage and CD+4/CD+8T in peripheral blood were significantly increased,and CD+8T lymphocyte percentage decreased significantly (P<0.01 or P<0.05). Conclusion Glycyrrhizic acid ointment's effect of treating chronic eczema may be related to regulating the balance of Th1 and Th2 and correcting the immune disorders.

Puma (Puma concolor) epididymal sperm morphometry / 亚洲男科学杂志(英文版)

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

Morphometric comparison by the ISAS® CASA-DNAf system of two techniques for the evaluation of DNA fragmentation in human spermatozoa / 亚洲男科学杂志(英文版)

DNA fragmentation has been shown to be one of the causes of male infertility, particularly related to repeated abortions, and different methods have been developed to analyze it. In the present study, two commercial kits based on the SCD technique (Halosperm ® and SDFA) were evaluated by the use of the DNA fragmentation module of the ISAS ® v1 CASA system. Seven semen samples from volunteers were analyzed. To compare the results between techniques, the Kruskal-Wallis test was used. Data were used for calculation of Principal Components (two PCs were obtained), and subsequent subpopulations were identified using the Halo, Halo/Core Ratio, and PC data. Results from both kits were significantly different (P < 0.001). In each case, four subpopulations were obtained, independently of the classification method used. The distribution of subpopulations differed depending on the kit used. From the PC data, a discriminant analysis matrix was obtained and a good a posteriori classification was obtained (97.1% for Halosperm and 96.6% for SDFA). The present results are the first approach on morphometric evaluation of DNA fragmentation from the SCD technique. This approach could be used for the future definition of a classification matrix surpassing the current subjective evaluation of this important sperm factor.

Spermiogram and sperm head morphometry assessed by multivariate cluster analysis results during adolescence (12-18 years) and the effect of varicocele / 亚洲男科学杂志(英文版)

This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age (mean 191, n = 87), who had reached oigarche by 12 years old, were recruited in the area of Barranquilla, Colombia. Morphometric analysis of sperm heads was performed with principal component (PC) and discriminant analysis. Combining seminal fluid and sperm parameters provided five PCs: two related to sperm morphometry, one to sperm motility, and two to seminal fluid components. Discriminant analysis on the morphometric results of varicocele and nonvaricocele groups did not provide a useful classification matrix. Of the semen-related PCs, the most explanatory (40%) was related to sperm motility. Two PCs, including sperm head elongation and size, were sufficient to evaluate sperm morphometric characteristics. Most of the morphometric variables were correlated with age, with an increase in size and decrease in the elongation of the sperm head. For head size, the entire sperm population could be divided into two morphometric subpopulations, SP1 and SP2, which did not change during adolescence. In general, for varicocele individuals, SP1 had larger and more elongated sperm heads than SP2, which had smaller and more elongated heads than in nonvaricocele men. In summary, sperm head morphometry assessed by CASA-Morph and multivariate cluster analysis provides a better comprehension of the ejaculate structure and possibly sperm function. Morphometric analysis provides much more information than data obtained from conventional semen analysis.

Células CD3+, CD4+, CD8+, CD3-CD16+CD56+ e CD19+ em sangue periférico de pacientes com hanseníase e indivíduos saudáveis

A hanseníase é uma doença granulomatosa, infecto-contagiosa causada pelo Mycobacterium leprae. Trata-se de uma infecção crônica com amplo espectro de respostas imunes celulares em humanos. Possui alto poder infectante e baixo poder patogênico. Este estudo tem como objetivo quantificar e comparar leucócitos e sub populações de linfócitos T totais (CD3+), T auxiliares (CD3+CD4+), T citotóxicos (CD3+CD8+), B (CD19+) e NK (CD3-CD16+CD56+) em sangue periférico de indivíduos com hanseníase e controles saudáveis.Os pacientes foram provenientesdo Centro de Dermatologia D. Libânia, Fortaleza-CE, Brasil. A determinaçãodo número de linfócitos em cada subpopulação foi realizada porcitometria de fluxo.Aanálise estatísticafoi realizadapelo programa Grap hPad Prism 5.0para Windows comsignificância estabelecida para valores de p<0,05.É um estudo do tipo caso controle de caráter observacional, realizado a partir da análise do sangue periférico de indivíduos com diagnóstico de hanseníase e de indivíduos saudáveis.A população de pacientes com hanseníase, sem tratamento foi composta de 15 pessoas. A população de controles saudáveis foi composta por 29 pessoas...

Leprosy is an infectious and granulomatous disease caused by Mycobacterium leprae. The aim of this study was to quantify and compare levels of leucocytes and lymphocyte subpopulations (CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3-CD16+CD56+) in peripheral blood ofpatients with leprosy and healthy controls. Patients were followed at Centro de Dermatologia D. Libânia, Fortaleza-CE, Brasil. Flow cytometry was used to determine numbers of lymphocytes. Statistical analisys was done with GraphPad Prism 5.0 software for windows. P values under 0.05 were considered siginificant.This was an observational case-control study. Fifteen leprosy patients without treatment were evaluated and 29 healthy individuals were included in control group...

Effects of intravenous immunoglobulin on immunity and cytokine levels in children with EV71 infection associated high-risk pulmonary hemorrhage / 实用医学杂志

Objective To investigate the influence of IVIG on immunologic function and cytokines levels in children with EV71 infection associated high-risk pulmonary hemorrhage. Methods According to the inclu-sion criteria , 64 children were enrolled and randomly divided into two groups: 39 cases in the IVIG treatment group and 25 cases in the general treatment group. The alternations of blood and immune cytokine markers before and after treatment were detected in the patients. Results (1) Before treatment, the peripheral blood T cells, TH and B cells in the IVIG group were higher than those in the general group , but the peripheral blood IgA was lower than that in the general group(P 0.05). Conclusion Disorders of cellular immunity and humoral immunity appeared in children with EV71 infection-re-lated high-risk pulmonary hemorrhage. It has clinical value to use IVIG timely to regulate the immune disorder.

Análisis genétioo de la población de gatos del norte y sur de cali, Colombia / Genetic Analysis of the Cat Population of North and South of Cali, Colombia

Los marcadores genéticos del pelaje y malformaciones óseas han permitido caracterizar el perfil genético de más de 400 poblaciones del gato doméstico alrededor del mundo. Hace 15 años se estableció dicho perfil en la ciudad de Cali (Colombia). En este estudio se determinó si el norte y sur de Cali se comportan como subpoblaciones y se comparó el perfil total con el estudio pasado. Se encontró una disminución de la frecuencia alélica de a (no-agouti) y d (dilution), pero un aumento en cinco, especialmente en l (longhair) y c s (siamese). Dichas diferencias pueden atribuirse a la selección humana de características más atractivas y por el flujo génico resultante del crecimiento demográfico de la ciudad, lo que explicaría también el primer reporte de los alelos inhibitor y ticked abyssinian. Se evaluó el equilibrio Hardy-Weinberg para el norte, sur y las dos zonas juntas, usando los loci white spotting y orange, encontrándose desequilibrio en este último para las tres zonas evaluadas debido a un déficit de heterocigotos. Norte y sur se dividieron en dos, y cada sub-muestra presentó equilibrio Hardy-Weinberg, aunque las diferencias en las frecuencias alélicas y heterocigosidades resaltaron microestructura geográfica y una relación entre tiempo de fundación del barrio y heterocigosidad. Norte y sur resultaron ser una población y no subpoblaciones (F ST= 0,0004, D= 0,0017), al igual que las nueve poblaciones colombianas con las que se comparó la presente ciudad. Se sugiere realizar un análisis microgeográfico de flujo génico y la definición de posibles colonias de gatos en Cali.

The coat genetic markers and skeleton abnormalities have allowed characterize the profile from more than 400 domestic cat populations around the world. 15 years ago, that profile was established in the city of Cali (Colombia). In this study it was determined if north and south of the city are subpopulations and it was compared the total profile against past study. A decrease in allele frequency of a (non-agouti) and d (dilution) was found, but an increase of five alleles was found, especially in l (long hair) and c s (siamese). These differences could be attributed to human selection of more attractive characteristics and gene flow resulting from demographic growth city, which would also explain the first report of inhibitor and ticked abyssinian alleles. Hardy-Weinberg equilibrium was evaluated for the north, south and both areas together, using white spotting and orange loci, determining disequilibrium in orange for the three evaluated areas due to a heterozygotes deficit. North and south were divided into two, each sub-sample showed Hardy-Weinberg equilibrium, although allele frequencies and heterozygosities highlighted microgeographic structure and a relationship between founding time of the neighborhood and heterozygosity. North and south are a single population and aren´t subpopulations (F ST= 0,0004, D= 0,0017), as well as nine Colombian populations with which this city was compared. It is suggested to make a microgeographical gene flow analysis and the definition of possible cat colonies in Cali.

Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation

To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFalpha level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 microg/m3, 112.3 microg/m3, and 2.3 microg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 microg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation.

Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: differentiation of subpopulations

Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98 percent inter-population, and 23 percent to 58 percent intra-population polymorphism. Nei's genetic distance between populations increased significantly with altitude (R² = 0.71) and geographic distance (R² = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei's gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R² = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D'2IS exceeded D'2ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool.

T lymphocyte sub-populations in malignant and tuberculous pleural effusion or ascites / 中华全科医师杂志

Objective To determine counts of T lymphocyte sub-populations in malignant and tuberculous pleural effusion or ascites and evaluate its significance in difierential diagnosis.Methods T lymphocyte sub-populations in pleural effusion or ascites and peripheral blood were determined in 30 patients with tuberculosis and 31 patients with cancer by flow cytometry.Concentrations of cytokines Th1 and Th2,γ-interferon(IFN-γ),interleukin-12(IL-12)and IL-4 in pleural effusion or ascites were measured by enzyme-linked immunosorbent assay(ELISA).Results Compared to that in peripheral blood,percentage of CD3+ and CD4+ T-celI counts were all higher in both malignant and tuberculous pleural effusion or ascites [(73±6)%and(67±20)%vs.(51±19)%and(48±14)%,P＜0.05].Although CD3+T-cells count was higher in tuberculous pleural effusions or ascites,no difference in ratio of CD3+ and CD4+/CD3+ and CD8+ T-cell counts was found between malignant and tuberculous pleural effusions or ascites.However,ratios of IFN-γ and IL-12 to IL-4 were higher in tuberculous pleural effusion or ascites(54±24 and 82±19vs.8±6 and 19±10,t=10.34 and 16.28,respectively,P＜0.01).Conclusions CD3+ and CD4+ Tcells can be aggregated in both malignant and tuberculous pleural effusions or ascites,80 nature (tuberculosis or malignancy)of pleural effusion or ascites can not be differentiated by CD4+ and/or CD8+ T-cell counts only,and determination of cytokines Th1 and Th2 can help their differentiation.