BST-1 as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

OBJECTIVES: Schizophrenia is a serious mental illness. The serum protein biomarkers of schizophrenia were explored using isobaric tags for relative and absolute quantitation (iTRAQ) technology. The underlying function of the identified protein biomarker was also investigated. METHODS: We first collected serum samples from 12 schizophrenia patients and 12 healthy control (HC) subjects, followed by global screening with iTRAQ and tandem mass spectrometry (LC-MS/MS). In total, 691 serum proteins were detected and eight proteins, including ZYX, OSCAR, TPM4, SDPR, BST1, ARGHDB, ITIH5 and SH3BGRL3, were selected for further specific validation with enzyme-linked immunosorbent assay (ELISA) on the serum samples from 52 schizophrenia patients and 50 HC subjects. RESULTS: Schizophrenia patients had significantly lower serum level of BST1 and higher ITIH5 level than the HC subjects did. Using the levels of BST1, ITIH5 and OSCAR combined with machine learning algorithm, we developed a prediction model of schizophrenia with an auROC value 0.78. Moreover, in vitro cell assay confirmed that BST1 significantly repressed neutrophil infiltration through endothelial layer, highlighted the anti-inflammation nature of BST1. CONCLUSIONS: Four novel protein markers (BST1, ITIH5, SDPR, and OSCAR) of schizophrenia were identified, and BST-1 could serve as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

Diagnosis of paroxysmal nocturnal hemoglobinuria with flowcytometry panels including CD157: Data from the real world.

BACKGROUND: Several studies have used CD157 in white blood cells with or without proaerolysin (fluorescein-labeled proaerolysin [FLAER])-based flow cytometry assays in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We designed a seven-color CD marker panel comprising FLAER, CD15, CD64, CD24, CD14, CD157, and CD45 to verify CD157's clinical applicability and diagnostic performance in a clinical setting. RESULTS: A total of 356 samples were tested. These included 43 PNH-positive samples and 313 PNH-negative samples. PNH clones confirmed by the CD157/FLAER combination were almost identical in size to the clones detected by the CD24/CD14/FLAER combination, and the accuracy of the CD157/FLAER combination was 100% in granulocytes and 99.7% in monocytes. Substitution of FLAER with CD157 resulted in 1.9% and 3.5% false-positives in granulocytes and monocytes, respectively. The accuracy was 98.3% and 96.9% in granulocytes and monocytes, respectively. Moreover, the loss of CD157 expression in granulocytes and monocytes was commonly observed in non-PNH patients. Some monocytes in non-PNH patients had weak expression of CD14 but normal expression of FLAER. In this study, PNH clones in granulocytes were always lower than those in matched monocytes. CONCLUSIONS: We performed the first prospective exploration of the clinical usefulness of FLAER and CD157 in simultaneously recognizing PNH clones in granulocytes and monocytes and verified the applicability of CD157 in substitute for both CD14 and CD24. In the conditions where FLAER is not available, substitution of FLAER with CD157 is acceptable for the identification of PNH clones under the premise of giving full attention to the potential for false-positives.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG-A) gene. STUDY DESIGN AND METHODS: In this study, the expression of a high number of GPI-APs on different subsets of peripheral blood (PB) cells from 14 PNH patients and their potential association with underlying genetic abnormalities has been analyzed. RESULTS: This study confirms the existence of variable patterns of expression of different GPI-APs on both major and minor PB-cell subsets from PNH patients. The size of the PNH clone within PB neutrophils and monocytes was systematically higher than that of other cell populations. Genetic changes were detected in the PIG-A gene in 5 of 13 cases analyzed. Interestingly, the reactivity for many GPI-APs was significantly higher on different subsets of normal PB cells from PNH patients than those observed on healthy volunteers. CONCLUSION: The best combination of markers for the diagnostic screening of PNH would include evaluation of CD14 on monocytes and of CD16 on neutrophils, although further analysis of CD55 and CD59 expression may contain additional clinically useful information. Clear association between the genetic changes detected in the PIG-A gene in 5 of 13 cases analyzed, and the phenotypic profile of PNH cells has not been found. Additionally, an abnormally higher expression of several GPI-APs among normal residual cells from PNH patients in comparison to healthy donors was observed, suggesting that factors other than the PIG-A mutation could determine the phenotypic profile of PB cells in PNH.

Decreased ADP-ribosyl cyclase activity in peripheral blood mononuclear cells from diabetic patients with nephropathy.

AIMS/HYPOTHESIS: ADP-ribosyl-cyclase activity (ADPRCA) of CD38 and other ectoenzymes mainly generate cyclic adenosine 5'diphosphate-(ADP-) ribose (cADPR) as a second messenger in various mammalian cells, including pancreatic beta cells and peripheral blood mononuclear cells (PBMCs). Since PBMCs contribute to the pathogenesis of diabetic nephropathy, ADPRCA of PBMCs could serve as a clinical prognostic marker for diabetic nephropathy. This study aimed to investigate the connection between ADPRCA in PBMCs and diabetic complications. METHODS: PBMCs from 60 diabetic patients (10 for type 1 and 50 for type 2) and 15 nondiabetic controls were fluorometrically measured for ADPRCA based on the conversion of nicotinamide guanine dinucleotide (NGD(+)) into cyclic GDP-ribose. RESULTS: ADPRCA negatively correlated with the level of HbA1c (P = .040, R(2) = .073), although ADPRCA showed no significant correlation with gender, age, BMI, blood pressure, level of fasting plasma glucose and lipid levels, as well as type, duration, or medication of diabetes. Interestingly, patients with nephropathy, but not other complications, presented significantly lower ADPRCA than those without nephropathy (P = .0198) and diabetes (P = .0332). ANCOVA analysis adjusted for HbA1c showed no significant correlation between ADPRCA and nephropathy. However, logistic regression analyses revealed that determinants for nephropathy were systolic blood pressure and ADPRCA, not HbA1c. CONCLUSION/INTERPRETATION: Decreased ADPRCA significantly correlated with diabetic nephropathy. ADPRCA in PBMCs would be an important marker associated with diabetic nephropathy.

The prognostic value of CD38 expression in chronic lymphocytic leukaemia patients studied prospectively at diagnosis: a single institute experience.

The purpose of this study was to assess in chronic lymphocytic leukaemia (CLL) patients the prevalence and clinical impact of CD38 expression, evaluated prospectively at disease presentation, and to verify whether this parameter changes over time. In 242 consecutive and untreated CLL patients, the percentage of CD38+ cases, according to the 7%, 20% and 30% cut-off points, was 21%, 17% and 14%, respectively. Using the 7% threshold, CD38 positivity correlated with male sex, intermediate and high-risk (Rai I-IV) disease, lower Hb and platelet levels, and higher lymphocyte count. Furthermore, patients with a CD38 expression>or=7% showed a significantly lower 3-year probability of treatment-free survival (TFS) than CD38- patients (P<0.0001). At multivariate analysis, CD38 expression remained significantly associated to TFS, together with stage, lymphocyte count and morphology. Also, in the 146 patients with stage 0 CLL a CD38 expression>or=7% identified a subgroup of patients with a significantly lower 3-year probability of TFS (P=0.0005). Furthermore, this parameter did not change in 30 of 31 (97%) re-evaluated patients. In conclusion, this study indicates that, when tested at diagnosis and on fresh material, a CD38 expression>or=7% is an important parameter for the identification of early CLL patients with more aggressive disease and that its expression remains stable over time.

Early proliferation of CCR5(+) CD38(+++) antigen-specific CD4(+) Th1 effector cells during primary HIV-1 infection.

We investigated whether HIV-1 antigen-specific CD4(+) T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38(+++)) and proliferating (Ki-67(+)) CD4(+) T cells that expressed CCR5(+), and were mostly T-cell intracellular antigen-1 (TIA-1)(+) perforin(+) granzyme B(+). Inthe same patient samples, CD4(+) T cells producing interferon (IFN)-gamma in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay-these cells were again predominantly CD38(+++), Ki-67(+), and TIA-(++), as well as Bcl-2(low). On average, 20% of the Gag-specific CD4(+) T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)(+). Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4(+) T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5(+)CD38(+++) CD4(+) T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5(+)CD4(+) cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.

NAD glycohydrolase activities and ADP-ribose uptake in erythrocytes from normal subjects and cancer patients.

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.

Serum levels of different forms of soluble CD38 antigen in burned patients.

The level of the total and dimeric (oligomeric) forms of soluble CD38 antigen (sCD38) has been determined by an ELISA sandwich method in serum from burned patients (n=18) and healthy volunteers (n = 25). The serum level of total sCD38 was insignificantly increased in patients at the stage of burn shock (135 +/- 10.8 U/ml, mean +/- S.E.M.) and significantly decreased between 4 and 14 postburn days in comparison with volunteers (69.5 +/- 10.8 U/ml versus 121 +/- 7.8 U/ml, P < 0.05). The serum level of soluble dimeric CD38 in burned patients was statistically lower than normal during all periods of observation (45.3 +/- 8.8 and 130 +/- 6.2 U/ml, respectively, P < 0.01). The relative number of CD38(+) lymphocytes was increased during the period of shock in comparison with healthy volunteers (21 +/- 1.6% versus 13 +/- 1.1%, P < 0.05). There were no correlations between number CD38(+) lymphocytes and total sCD38 or dimeric sCD38 serum levels. These data suggest that the mCD38 expression and serum level of total sCD38 are a markers the early postburn lymphocytes activation. The decrease of dimeric sCD38 level can reflect its dissociation to monomeric form in burned patients.

Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage.

OBJECTIVE: To identify genes involved in megakaryopoiesis, high-density oligonucleotide microarrays were used to compare transcript profiles from undifferentiated CD34+CD38lo cells and culture-derived megakaryocytes (MKs). MATERIALS AND METHODS: Megakaryocyte differentiation was achieved in vitro by inducing primary human CD34+CD38lo cells in serum-deprived media supplemented with the cytokine combination of interleukin-3, interleukin-6, stem cell factor, and thrombopoietin for 10 days. Three replicate microarray experiments were performed using hematopoietic cells isolated from three different organ donors and high-density oligonucleotide microarrays. RESULTS: Analysis of gene array data resulted in 304 differentially expressed genes (p < or = 0.001, fold change > or = 3). A third of the 25 most highly up-regulated genes were known to participate in hemostasis (z = 6.75), and no genes known to be associated with MKs were among the down-regulated genes. We also found a large proportion of up-regulated transcripts in gene ontology categories of adhesion and receptor activity (85%) and signal transduction activity (68%). At the same time, 70% of genes within transcription factor functions were down-regulated. Confirmatory studies indicated that the array results correlated with mRNA and protein expression levels in primary MKs. CONCLUSION: This study provides a global expression profile of human MKs and a list of novel and previously uncharacterized candidate genes that are important components of megakaryopoiesis.

Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations.

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.

Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis.

The molecular mechanisms that regulate asymmetric divisions of hematopoietic progenitor cells (HPCs) are not yet understood. The slow-dividing fraction (SDF) of HPCs is associated with primitive function and self-renewal, whereas the fast-dividing fraction (FDF) predominantly proceeds to differentiation. CD34+/CD38- cells of human umbilical cord blood were separated into the SDF and FDF. Genomewide gene expression analysis of these populations was determined using the newly developed Human Transcriptome Microarray containing 51 145 cDNA clones of the Unigene Set-RZPD3. In addition, gene expression profiles of CD34+/CD38- cells were compared with those of CD34+/CD38+ cells. Among the genes showing the highest expression levels in the SDF were the following: CD133, ERG, cyclin G2, MDR1, osteopontin, CLQR1, IFI16, JAK3, FZD6, and HOXA9, a pattern compatible with their primitive function and self-renewal capacity. Furthermore, morphologic differences between the SDF and FDF were determined. Cells in the SDF have more membrane protrusions and CD133 is located on these lamellipodia. The majority of cells in the SDF are rhodamine-123dull. These results provide molecular evidence that the SDF is associated with primitive function and serves as basis for a detailed understanding of asymmetric division of stem cells.

Virological and immunological outcomes in HIV-1-infected Chinese patients treated with a combination of Efavirenz and Indinavir for 48 weeks.

BACKGROUND: The incidence of HIV-1-related infection diseases and the mortality of AIDS have dramatically decreased since highly active antiretroviral therapy began to be used clinically in China in 1999. And we initiated a second clinical trial using a combination of Efavirenz and Indinavir to observe the effects of the immunoreaction. METHODS: Twenty patients with laboratory-confirmed chronic HIV-1 infection were recruited. Blood samples were collected initially and during the weeks after initiation of treatment. Within 48 hours of blood sampling, peripheral blood plasma and mononuclear cells were separated using routine methods. HIV-1 viral load was measured in thawed plasma samples. Within 48 hours of peripheral blood sampling, CD4(+) and CD8(+) T cell subsets were enumerated. RESULTS: The drug regimen was efficient in reducing HIV-1 plasma viral load and increasing total CD4(+) T cell counts. The percentage of CD4(+) and CD8(+) T cell subsets expressing CD38 and HLA-DR activation markers was positively correlated with plasma viral load and tended to normalize. CONCLUSIONS: The combination of Efavirenz and Indinavir was generally well tolerated and efficient at reducing HIV-1 RNA. Furthermore, the treatment improved the immunological function.

Immunophenotyping of blood lymphocytes at birth, during childhood, and during adulthood in HIV-1-uninfected Ethiopians.

To obtain more insight into blood lymphocyte subpopulations of Ethiopians, we studied the immunologic profile of children and neonates and compared these data with those obtained from adults. Peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were collected from 137 HIV-1-uninfected subjects aged 0 (cord blood) up to 40 years. Lymphocyte subsets (T, B, and NK cells, CD4+ and CD8+ T cells) were determined and T cell activation (CD38 and HLA-DR) and differentiation (CD45RO and CD27) markers were measured on CD4+ and CD8+ T cells. The absolute number and percentage values of most lymphocyte subpopulations differed substantially with age. Neonates and children were found to have significantly higher CD4+ T cell counts compared to adults. The median absolute CD4 count at birth was comparable to those reported for Caucasians. At birth 97% of the CD4+ T cells were naîve and this proportion significantly declined to 14.2% during adulthood. In addition, activation of both CD4+ and CD8+ T cells, as determined by the double expression of HLA-DR and CD38, was observed in children under the age of 16 and adults, but not in neonates. A more differentiated phenotype (CD27-) was observed in adults compared to children for both CD4+ and CD8+ T cells. The immune alterations including the remarkably low CD4 count with highly depleted naîve phenotype and a persistently activated immune system seen in adult Ethiopians are not apparent at birth, but rather develop over time.

Changes in levels of immune activation and reconstitution markers among HIV-1-infected Africans receiving antiretroviral therapy.

OBJECTIVE: To describe changes in immune activation and reconstitution markers among HIV-1-infected patients receiving antiretroviral therapy (ART) in Abidjan, Côte d'Ivoire. METHODS: Between November 1998 and February 2001, we analyzed changes in immune activation and reconstitution markers among 52 patients. Good virologic responders (n = 26) were defined as those who had suppressed and maintained plasma viral load (VL) below the detection limit of the assay for at least 12 months. Poor virologic responders (n = 26) were defined as those with a detectable VL at 6 and 12 months after beginning ART. RESULTS: Of the 26 good virologic responders, 20 (77%) were on highly active antiretroviral therapy (HAART) compared with one (4%) of the poor responders. Among the 26 good responders, baseline median levels of CD38+CD8+ T cells were elevated, but had decreased significantly at 6 months (P < 0.001) and at 12 months of therapy (P < 0.001). Median levels of HLA-DR+CD8+ T cells also decreased from baseline at 6 months (P < 0.001) and at 12 months of therapy (P < 0.001). Levels of CD62L+CD4+ T cells increased steadily during the 6 and 12 months of therapy and reached levels observed among HIV-negative blood donors (P = 0.07). Among the 26 poor responders, median levels of CD38+CD8+ T cells decreased significantly at 12 months of therapy (P = 0.006), but were higher than levels in blood donors (P = 0.005). Levels of HLA-DR+CD8+ T cells decreased significantly at 12 months of therapy (P < 0.001). Levels of CD62L+CD4+ decreased over time. CONCLUSION: Our results suggest that HAART can be successfully used in African populations with elevated baseline immune activation markers.

[Prognositc value of CD38 cell surface marker in chronic lymphocytic leukemia]. / CD38 sejtfelszíni marker prognosztikai jelentósége krónikus lymphoid leukaemiában.

CD38 is expressed on the surface of leukemic cells in a significant percentage of patients with B-cell chronic lymphocytic leukaemia (CLL). From the literature it is known that CD38 expression has prognostic value in CLL, suggesting an association between CD38 expression and the mutational status of IgV genes. Peripheral blood samples from 82 patients with CLL were analyzed by flow cytometry for CD38 expression on CD19+ leukemic cells. CD38 was expressed in 30% or more of leukemic cells in 26 patients (patients with 30% or more B cells coexpressing CD19/CD38 were considered positive). Seven of the 26 patients with high CD38 expression and eight of the 56 patients with low CD38 expression had advanced-stage disease (RAI III-IV). Higher than 4 mg/l levels of beta 2-microglobulin was measured in the serum of nine of 26 CD38+ and seven of 56 CD38- patients. Our analyses showed that the high CD38 expression is associated with other risk factors, identifying an aggressive disease, which require treatment. It will be important to conduct further studies to establish the prognostic value of the high CD38 expression in early-stage disease.

Relative importance of CD38 expression over myeloid-associated markers expression in predicting the clinical course of B-CLL patients.

Expression of CD38 or myeloid-associated markers has been reported to be important in predicting prognosis in B-cell chronic lymphocytic leukemia (B-CLL) in separate studies but the impact of combining these markers on prognosis has not been examined. The current study aimed to evaluate the relative contribution of expression of CD38 and/or myeloid-associated markers (CD11b, CD13, CD15 and CD33) by flow cytometry (FCM) on the clinical course of 24 B-CLL patients. B-CLL patients with high levels of CD38 expression, defined as greater than or equal to 30% of neoplastic lymphocytes expressing CD38, had a significantly poorer OS than those with low levels of CD38 expression (54% cumulative survival: 51 months vs. 103 months, Kaplan-Meier survival analysis, p < 0.005, Logrank test). High levels of expression of myeloid-associated markers showed no statistically significant impact on OS in these patients. Ten of 11 patients (91%) with high levels of CD38 expression required chemotherapy. In contrast, only 5 of 13 patients (38%) with low levels of CD38 expression required chemotherapy (p < 0.009, Chi Square). There was no significant difference in the requirement for chemotherapy between patients with high levels of expression of myeloid-associated marker and those without (5/8 or 63% vs. 10/16 or 63%). Thus, our results suggest that CD38 is superior to myeloid-associated markers in predicting the prognosis of patients with B-CLL. Further studies with a larger sample size are indicated to confirm our observation.

Determination of laboratory reference ranges for lymphocyte immunophenotype subsets in healthy adult Japanese using flow cytometry.

This study represents a comprehensive evaluation of normative values for lymphocyte immunophenotype subsets using flow cytometry techniques in a Japanese population. Lymphocyte reference ranges were determined for percentage and absolute count of T, B, and NK cells in healthy adult Japanese using an extensive two-color immunophenotyping panel and consistently applied quality control methodology. Reference values were also determined for activation markers on CD3+ lymphocytes CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+. Differences in age and gender were observed for specific lymphocyte subsets. Comparison of the Japanese study with a Thai multi-center study that used similar methodology also demonstrated ethnic differences in lymphocyte reference ranges. The results in this study strongly suggest that reference values derived from studies in one population may not be applied to another population even when similar protocols for reagents, instruments and procedures are used although such studies do appear useful for epidemiological comparisons.