Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.     

Human pluripotent and progenitor cells display cell surface cluster differentiation markers CD10, CD13, CD56, and MHC class-I.

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality. A wide variety of transplants, congenital malformations, elective surgeries, and genetic disorders have the potential for treatment with autologous stem cells as a source of HLA-matched donor tissue. Our current research is aimed at characterizing cell surface cluster differentiation (CD) markers on human progenitor and pluripotent cells to aid in isolating comparatively purified populations of these cells. This study examined human pluripotent and progenitor cells isolated from fetal, mature, and geriatric individuals for the possible presence of 15 CD markers. The response to insulin and dexamethasone revealed that the cell isolates were composed of lineage-committed progenitor cells and lineage-uncommitted pluripotent cells. Flow cytometry showed cell populations positive for CD10, CD13, CD56, and MHC Class-I markers and negative for CD3, CD5, CD7, CD11b, CD14, CD15, CD16, CD19, CD25, CD45, and CD65 markers. Northern analysis revealed that CD13 and CD56 were actively transcribed at time of cell harvest. We report the first identification of CD10, CD13, CD56, and MHC Class-I cell surface antigens on these human cells.     

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.     

Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by pancytopenia, dysplastic hematopoiesis, and a propensity to leukemic transformation. Increased apoptosis has been noted in MDS as a possible explanation for ineffective hematopoiesis, with lower levels in progression to and in de novo acute leukemia. Apoptosis can be measured by binding of Annexin V to exposed membrane phosphatidylserine. We postulated that the apoptotic index would aid in the differential diagnosis of MDS versus other hematopoietic diseases. We examined 33 bone marrow aspirates suspected of hematopoietic malignancy for apoptotic index by Annexin V analysis using a Becton Dickinson FACStar+ flow cytometer. The apoptotic index was expressed as the percentage of Annexin V-positive cells divided by total mononuclear cells in the gate. By standard morphologic analysis, 16 cases were diagnosed as MDS (9 refractory anemia [RA], 2 refractory anemia with ringed sideroblasts [RARS], 1 refractory anemia with excess of blasts [RAEB], 3 chronic myelomonocytic leukemia [CMML], and 1 unclassified), 11 as acute leukemia (AL), 6 as myeloproliferative disorders (MPD). Eight cases (uninvolved marrow of five patients with lymphoproliferative disorders [LPD], one patient with multiple myeloma, and two patients with anemia of chronic disease) served as nonneoplastic controls. A higher degree of apoptosis was observed in MDS (mean = 44.7%; range = 29.5--60%) compared with MPD (mean = 8.2%; range = 2.3--15.4%), AL (mean = 16.1%; range = 5.1--29.4%), and control marrow samples (mean = 11.6%; range = 1.5--21%). Additionally, the apoptotic index was significantly higher in MDS compared with MPD (P < 0.0001). In conclusion, a high apoptotic index occurs in MDS, supporting previous reports and suggesting that Annexin V analysis can be used as an adjunct in the diagnosis of MDS versus MPD. This would be particularly useful for the often-difficult distinction between early MDS and early MPD cases with equivocal morphology.     

The Effect of Fetal and Early Postnatal Iron Deficiency on Iron Metabolism in Adult Rats

Undernutrition during pregnancy and/or lactation plays an important role on the overall health of offspring later in life. Using a rodent model, the present study was conducted to examine the effect of fetal and early postnatal iron deficiency on iron metabolism in adult animals. Rats were treated with three stages of low or normal iron diets from gestation until the end of the study. During the first stage (4?weeks prior to 3?weeks after pregnancy, total 7?weeks), two groups of adult females (dams) were fed with either a low-iron (7.4?mg iron/kg, group LD) or control-iron (274?mg/kg, group CD) diet. During the second stage (from 3 to 13?weeks of age, total 10?weeks), all pups from stage 1 (both the LD and CD groups) were placed on a control-iron diet for 10?weeks (groups LD?CCD and CD?CCD, respectively). During the third stage (from 13 to 29?weeks of age, total 16?weeks), both LD?CCD and CD?CCD groups from stage 2 were fed with a low-iron (named LD?CCD?CLD and CD?CCD?CLD groups, respectively). We found that the live birth rate of the offspring of the LD dams (84.7?%) was significantly lower than that of the CD dams (95.4?%). During stage 2, the mean body weight of the LD?CCD male or LD?CCD female rats exceeded the CD?CCD male rats (p?<?0.05). Compared with the CD?CCD?CLD rats, the LD?CCD?CLD rats had significantly increased total iron binding capacity, and higher levels of transferrin, serum erythropoietin (EPO), renal EPO mRNA, duodenal divalent metal transporter-1, and renal transferrin receptors. These findings indicate that rats with an early-life experience of iron deficiency (during pregnancy and the nursing period) can develop stronger iron absorption capabilities in adulthood.     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. METHODS: Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.     

Selenium levels in men with liver disease in Hungary

ProjectWe studied the relationship between selenium (Se) levels and chronic liver disease (CLD) severity and the association between socioeconomic and lifestyle factors and serum Se levels.ProcedureWe performed a case–control study in Hungarian men, examining 281 patients with CLD and 778 controls. Liver function was evaluated using biochemical markers, and liver disease was verified with physical examination and blood tests. Linear regression analysis was performed to study the association of serum Se level with biochemical markers in cases and controls. In control participants we examined the relationship between Se levels and age, financial status, education, alcohol consumption, cigarette smoking, type of fat used for cooking and body mass index.ResultsSerum Se levels were lower in cases (median 0.87 μmol/L (IQR: 0.77–1.03)) than in controls (median 1.08 μmol/L (IQR: 0.97–1.19)). In controls, increases in bilirubin and glutamic-oxaloacetic transaminase (GOT) were associated with decreases in Se levels. In patients with CLD, a statistically significant relationship was found between serum Se and the GOT/GPT ratio, albumin and bilirubin. Younger, better-educated controls had significantly higher, and regular smokers and heavy drinkers had significantly lower Se levels. The use of vegetable oil/fat was also associated with higher Se levels. Se level was associated with the severity of liver injury in people even in patients who did not exhibit signs and symptoms of CLD.ConclusionsSerum Se level is strongly associated with the severity of liver damage in people with CLD from the early stage on.     

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, and remains incurable in many cases. Developing more efficient immunotherapy strategies will require better understanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers. Apart from their role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells have been recently revealed as one of the key players in the immune responses against tumors. The objective of this study was to evaluate the frequency of CD3(+)/CD16(+)CD56(+) cells in the peripheral blood of 28 diffuse large B-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentages of CD3(+)/CD16(+)CD56(+) were significantly lower in patients with DLBCL compared to healthy donors (7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts. The frequency and the absolute numbers of CD3(+)/CD16(+)CD56(+) cells were lower in advanced clinical stages than in earlier ones. The median percentage of CD3(+)/CD16(+)CD56(+) cells in patients in Ann Arbor stages 1-2 was 5.55% vs. 3.15% in stages 3-4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p = = 0.02). The percentage and absolute numbers of CD3(+)/CD16(+)CD56(+) cells were significantly higher in DL -BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04; 0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3(+)/CD16(+)CD56(+) cells correlated adversely with serum lactate dehydrogenase (R= -445; p 〈 0.05) which might influence NKT count. These figures suggest a relationship between higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains to be explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of their suppression by the tumor cells, or their migration to affected lymph nodes or organs.     

The IL-12 signature: NK cell terminal CD56+high stage and effector functions

We report that human peripheral NK cells expressing high CD56 levels (CD56(+high)) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56(+low) NK cells. CD56(+high) NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56(+)) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56(+high) than on CD56(+low) NK cells. Consistent with their activated phenotype, many CD56(+high) NK cells are cycling and mediate heightened effector functions (proliferation, IFN-gamma and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56(+low) NK cells all markers constitutively expressed on the CD56(+high) NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56(-/+low) NK cells acquire a CD56(+high) NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.     

CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells

Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56^dimCD16^bright make up 90% circulating NK cells, whereas CD56^brightCD16^-/dim comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56^bright NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-γ and TNF-α and notably IL-12.     

Clinical immunological disorders in children from various observation cohorts exposed to radiation factor during various stages of oncogenesis

The immune status disorders and features depending on the radiation impact type in various cohorts of radiation observations long after the Chernobyl (CNPP) disaster and the possible role of these disorders in development of chronic somatic pathology in children are shown. Lymphocyte depletion, T-cell immunity component disorders in the form of cell contraction with CD3, CD4, CD8 markers and the B-cell immunity component disorders in the form of reducing the quantity of CD10, CD23 marker cells were observed in children subject to combined chronic irradiation by 131I, 137Cs, 90Sr radionuclides. The descendants of irradiated parents (the 1st generation; children of the Chernobyl accident consequences liquidators, children of the citizens of radiation contaminated territories with various 137Cs levels) had immunity disorders of different type. A change in the total amount of NK-cells (CD16(+)-lymphocytes) is the general sign for all radiation risk groups; however, people subject to direct radiation impact demonstrated reduction of the antitumor protection potency, whereas descendants of irradiated ones demonstrated its activation with typically increasing number of CD16(+)-lymphocytes. In all radiation risk groups, a tendency to reduction of a number of cells involved in the leukocytal activation with the "pluripotential activation" marker (CD38 marker cells), proliferating cells (CD71 marker cells) and the increase of relative amount of cells with apoptosis marker (CD95(+)-lymphocytes). Immune disorder markers under the radiation impact in various cohorts of children's observation are suggested: antigens: CD4, CD8, CD10, CD23, CD16, CD38, CB71, CD95.     

Effects of Colesevelam,Rosiglitazone, or Sitagliptin on Glycemic Control and Lipid Profile in Patients with Type 2 Diabetes Mellitus Inadequately Controlled by Metformin Monotherapy

ObjectiveTo evaluate the glycemic effect of colesevelam, rosiglitazone, or sitagliptin when added to metformin monotherapy in patients with type 2 diabetes mellitus (DM) and to examine the effects of these antidiabetes agents on lipid and lipoprotein levels.MethodsThis 16-week, open-label pilot study conducted between May 2007 and April 2008 at 20 sites in the United States, 7 sites in Mexico, and 6 sites in Colombia, enrolled adults with inadequately controlled type 2 DM (glycated hemoglobin [HbA_1c], 7.0%-10.0%) on a stable metformin regimen (1500-2550 mg daily for ≥ 3 months). At Week 0, participants were randomly assigned 1:1:1 to open-label colesevelam hydrochloride, 3.75 g daily; openlabel rosiglitazone maleate, 4 mg daily; or open-label sitagliptin phosphate, 100 mg daily, in addition to existing metformin therapy. The primary efficacy variable was the change in HbA_1c from baseline to Week 16 with last (postbaseline) observation carried forward.ResultsIn total, 169 participants were randomly assigned to treatment groups (colesevelam, n = 57; rosiglitazone, n = 56; and sitagliptin, n = 56), and 141 participants (83.4%) completed the study. Least-squares mean reductions in HbA_1c from baseline were observed in all groups at Week 16 last observation carried forward (colesevelam, -0.3% [P <.031]; rosiglitazone: -0.6% [P <.001]; sitagliptin: -0.4% [P <.009]) At study end, 10 of 56 participants (17.9%) in the colesevelam group, 19 of 54 (35.2%) in the rosiglitazone group, and 15 of 55 (27.3%) in the sitagliptin group achieved HbA_1c < 7.0%. Colesevelam significantly reduced mean low-density lipoprotein (LDL)-cholesterol levels relative to baseline (11.6%), whereas levels were significantly increased with rosiglitazone and sitagliptin at Week 16 last observation carried forward (7.8% and 7.7%, respectively). Twenty-two of 52 participants (42.3%) in the colesevelam group, 12 of 51 (23.5%) in the rosiglitazone group, and 13 of 53 (24.5%) in the sitagliptin group achieved LDL cholesterol < 100 mg/dL at Week 16 last observation carried forward.ConclusionAll 3 antidiabetes agents significantly improved glycemic control, but only colesevelam also significantly reduced LDL-cholesterol levels in patients with type 2 DM. (Endocr Pract. 2010;16:53-63)     

Stage-specific induction of terminal deoxynucleotidyl transferase in a T-lymphoid line upon coculture with a thymic stromal line

We previously reported an in vitro T-cell differentiation system in which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T. L4 cells in L4—St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1⁻CD4⁻ phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time. The magnitude of TdT RNA induction was maximal for cell lines with the least mature differentiation phenotype (15#4T and L4: Thy-1⁻CD4⁻) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1⁺CD4⁺). TdT protein was undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay. In contrast to TdT, RAG-1 expression was suppressed by coculture with St3 cells for 15#4T and also more mature subclones, indicating regulation by a mechanism independent from TdT. The ordered induction of TdT, Thy-1, and CD4, as well as regulation of RAG-1 in the 15#4T-St3 system, supports the conclusion that this in vitro system is a valuable model for characterizing regulation of these markers in normal thymocytes.     

Comparative studies of CD3- and CD3+ CD56+ cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression.

Both CD3- and CD3+ CD56+ effector cells can mediate non-MHC-restricted lysis in the absence of activation. Previous studies have shown that both of these subsets can be augmented with IL-2. In the present study, we have examined further the phenotypic markers expressed on these cells as well as the functional capacities of these subsets, including LAK activity, cytokine expression, and pore-forming protein (PFP) production. In addition, these populations were analyzed for clonality by Southern blot analysis of the T cell receptor beta chain gene constant region. The CD3-, CD56+ and CD3+, CD56+ lymphocytes were quite similar in their phenotypic markers, although the CD3+, CD56+ lymphocytes lacked high levels of IL-2 receptor beta chain and did not express CD16. The CD3+, CD56+ lymphocytes mediated non-MHC-restricted lysis, but failed to express LAK activity or be induced by IL-2 to secrete IFN gamma, a characteristic of the CD3-, CD56+ lymphocytes. The T cell receptor beta chain gene pattern of the CD3+, CD56+ lymphocytes was characteristic of a polyclonal cell population. Of interest, both populations of cells appeared morphologically to be large granular lymphocytes that contain PFP in their cytoplasmic granules. Therefore these CD56+ subsets provide a new model to study several questions related to non-MHC-restricted target cell lysis, including the identification of novel receptors involved in target cell recognition and/or triggering as well as the biochemical pathways implicated in cellular lysis.     

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.     

Lymphocyte surface marker genes in fugu

At present, much of the fish adaptive immune system remains unknown due to the paucity of marker-specific reagents (e.g. antibodies) to identify immune cells. The genomic sequence of Takifugu rubripes (fugu) represents an important resource to facilitate the identification of lymphocyte-related genes. Here, we review the recent works on B-lymphocyte markers, the heavy (H) chains of IgM and IgD, and Ig light chain, and T-cell marker gene homologues, CD3ε, CD3γ/δ, CD4, and CD8α in a single fish species, fugu. Expressions of these B- and T-lymphocyte markers homologues in peripheral blood leukocytes and other lymphoid tissues of fugu suggest that these molecules in fish would be available as lymphocyte markers. These findings will lead us to develop reliable reagents for the identification of lymphocyte subpopulations. Fugu holds great promise as one of the model organisms for studies of development and evolution of adaptive and innate immunity in vertebrates.     

Assessing genetic diversity and differentiation in Portuguese coarse-wool sheep breeds with microsatellite markers

Population structure and genetic diversity in the Portuguese native breeds of sheep Algarvia (AL), Badana (BA), Galega Bragançana (GB), Galega Mirandesa (GM), Mondegueira (MO) and Churra da Terra Quente (TQ), as well as the exotic Assaf (AS), were analyzed by typing 25 microsatellite markers in 210 individuals. The markers used exhibited high levels of polymorphism, with means for total and effective number of alleles per locus of 13.0 and 4.2, respectively, and an expected heterozygosity of 0.72 across loci. The mean number of alleles per locus and expected heterozygosity were highest in GM and GB, and lowest in AS. Exclusive alleles were found in 10 of the 25 markers analysed, mostly in the AS breed. The proportion of loci which were not in Hardy–Weinberg equilibrium in each breed ranged between 0.12 (GB) and 0.40 (AL and GM), mostly due to a lower than expected number of heterozygotes in those loci. All breeds showed a significant deficit in heterozygosity, which was more pronounced in GM (F_IS = 0.113) and BA (F_IS = 0.103), suggesting that inbreeding might be a major concern in these breeds. The analysis of relationships among breeds, assessed by different methods, indicates that AS and AL are the more distanced breeds relative to the others, while the closest relationships were observed between TQ with MO and GM with GB. The estimated F_ST indicates that only 0.049 of the total genetic variability can be attributed to differences among breeds, and this ratio dropped to 0.029 when only the native breeds were considered. The analysis of individual distances based on allele-sharing indicates that only AS and AL had a tendency for animals of the same breed to cluster together, while for the other breeds there was overlapping among breeds. The results of this study confirm that native breeds of sheep represent an important reservoir of genetic diversity, even though the level of differentiation among closely located breeds tends to be rather small. For several of the breeds analyzed, the levels of inbreeding currently observed cause some apprehension, and recommend the establishment of appropriate conservation strategies, aimed at minimizing inbreeding to avoid further losses of genetic diversity.     

185例儿童急性白血病流式细胞术免疫分型研究

目的：探讨儿童急性白血病流式细胞术免疫分型的意义。方法：采用流式细胞术三色荧光标记技术和CD45／SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果：流式细胞术免疫分型和FAB分型的符合率为89．19％。185例儿童急性白血病中,ALL为121例,占AL的65．41％,B—ALL为113例,主要表达B系的CD19（99．12％）、CD22（98．13％）、CD79a（96．19％）、CD10（86．73％）。T—ALL占8例;主要表达CD5（100％）、CD7（100％）、cCD3（100％）、CD8（87．5％）。AML为47例,占25．41％,主要表达CD33（93．62％）、CD15（78．72％）、CD64（76．6％）、MPO（76．6％）、CD13（74．47％）。在B—ALL,AML,T—ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9．19％,其中B／M为9例,T／B为5例,T／M为3例。My＋-ALL为54例,占ALL的44．63％,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly＋-AML为18例,占AML的38．30％,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67．57％,HLA—DR的表达率为85．41％,CD38的表达率为80．59％,TdT的表达率为62．59％。结论：流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率。有必要进一步加强流式细胞术免疫分型的标准化工作。     

Human Papillomavirus (HPV) 16 E6 seropositivity is elevated in subjects with oral HPV16 infection

IntroductionHuman Papillomavirus (HPV) 16 E6 serum antibodies are common in people with HPV-related oropharyngeal cancers (HPV-OPC), but not the general population. We explored HPV16 seroprevalence in people with and without oral HPV16 infection, the cause of HPV-OPC.MethodsOral rinse samples were collected semiannually and tested for 36 types of HPV DNA by PCR. HPV16 E6 serum antibodies were tested at the visit of first oral HPV detection in participants with prevalent (n = 54), or incident (n = 39) oral HPV16 DNA; or at baseline in matched participants with no oral HPV16 DNA (n = 155) using multiplex serology assay. Predictors of seropositivity were examined using logistic regression.ResultsHPV16 E6 seropositivity (7.5% vs 0.7%; p = 0.005) but not seropositivity to the other HPV16 antigens, was significantly more common in those with than without oral HPV16 infection. There were only 8 HPV16 E6 seropositive participants, but oral HPV16 DNA remained a strong predictor of E6 seropositivity after adjustment for other risk factors (aOR = 14.6 95%CI, 1.7–122.5). Seroprevalence was similar in those with prevalent (7.4%; 4/54), and incident (7.7%; 3/39) oral HPV16 infection (p = 1.00). E6 seroprevalence was associated with reduced oral HPV16 clearance, but was not statistically significant (HR = 0.65 95% CI, 0.16–2.70).Seropositive participants were primarily male (87.5%), HIV-positive (75.0%; median CD4 cell-count of 840) and had oral HPV16 DNA (87.5%). History of an HPV-related cancer (0/8) or HPV-related anogenital dysplasia (1/8) was rare, and 4 participants had recent screening showing no anogenital dysplasia.DiscussionHPV16 E6 seropositivity was higher among people with than without oral HPV16 infection, despite no known anogenital disease in these participants.