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Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50-60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.

Juvenile myelomonocytic leukemia (JMML), a rare myelodysplastic/myeloproliferative neoplasm of early childhood, is characterized by clonal growth of RAS signaling addicted stem cells. JMML subtypes are defined by specific RAS pathway mutations and display distinct gene, microRNA (miRNA) and long non-coding RNA expression profiles. Here we zoom in on circular RNAs (circRNAs), molecules that, when abnormally expressed, may participate in malignant deviation of cellular processes. CirComPara software was used to annotate and quantify circRNAs in RNA-seq data of a "discovery cohort" comprising 19 JMML patients and 3 healthy donors (HD). In an independent set of 12 JMML patients and 6 HD, expression of 27 circRNAs was analyzed by qRT-PCR. CircRNA-miRNA-gene networks were reconstructed using circRNA function prediction and gene expression data. We identified 119 circRNAs dysregulated in JMML and 59 genes showing an imbalance of the circular and linear products. Our data indicated also circRNA expression differences among molecular subgroups of JMML. Validation of a set of deregulated circRNAs in an independent cohort of JMML patients confirmed the down-regulation of circOXNAD1 and circATM, and a marked up-regulation of circLYN, circAFF2, and circMCTP1. A new finding in JMML links up-regulated circMCTP1 with known tumor suppressor miRNAs. This and other predicted interactions with miRNAs connect dysregulated circRNAs to regulatory networks. In conclusion, this study provides insight into the circRNAome of JMML and paves the path to elucidate new molecular disease mechanisms putting forward circMCTP1 up-regulation as a robust example., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dal Molin, Hofmans, Gaffo, Buratin, Cavé, Flotho, de Haas, Niemeyer, Stary, Van Vlierberghe, Philippé, De Moerloose, te Kronnie, Bresolin, Lammens and Bortoluzzi.)

Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results., Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples., Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I., Conclusion: Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles., (Copyright © 2020 Linskens, Diks, Neirinck, Perez-Andres, De Maertelaere, Berkowska, Kerre, Hofmans, Orfao, van Dongen, Haerynck, Philippé and Bonroy.)

One of the most important problems in controlling malaria is the limited access to effective and accurate diagnosis of malaria parasitemia. In the Democratic Republic of Congo (DRC), malaria is one of the leading causes of morbidity and mortality. The purpose of this study was to assess the prevalence of anemia and the relationship with asymptomatic submicroscopic Plasmodium infection. A cross-sectional study was carried out among 1,088 apparently healthy children aged between 6 and 59 months selected at random in the health zone of Miti Murhesa in South Kivu/DRC. Capillary blood was obtained for hemoglobin (Hb) concentration measurement by Hemocue ® Hb 301. Malaria detection was performed by microscopy and the loop-mediated isothermal amplification (LAMP) assay. Anemia was defined as Hb < 11g/dL. We applied the chi-square test for comparisons, and multiple logistic regression was used to identify the risk factors for anemia and submicroscopic Plasmodium infection. The prevalence of anemia was 39.6%, and the prevalence of parasitemia was 15.9% and 34.0% using microscopy and LAMP test, respectively. Submicroscopic Plasmodium infection was found in 22.3% of the children. The independent risk factors for anemia are Plasmodium infection, children younger than 24 months, low middle-upper arm circumference, and history of illness two weeks before. Otherwise, children with submicroscopic malaria infection have a significantly increased risk for anemia, with a need of transfusion. The prevalence of malaria infection was underestimated, when microscopy was used to diagnose malaria. Children with low parasitemia detected by LAMP but not by microscopy showed a significantly increased prevalence of anemia.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML., (Copyright© 2020 Ferrata Storti Foundation.)

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 ( n = 4, two of them presented with Omenn syndrome), IL2RG ( n = 4, one of them with confirmed maternal engraftment), NHEJ1 ( n = 1), CD3E ( n = 1), ADA ( n = 1), JAK3 ( n = 3, two of them with maternal engraftment) and DCLRE1C ( n = 1) and 11 other PID patients had diverse molecular defects [ ZAP70 ( n = 1), WAS ( n = 2), PNP ( n = 1), FOXP3 ( n = 1), del22q11.2 (DiGeorge n = 4), CDC42 ( n = 1) and FAS ( n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs)., (Copyright © 2020 Kalina, Bakardjieva, Blom, Perez-Andres, Barendregt, Kanderová, Bonroy, Philippé, Blanco, Pico-Knijnenburg, Paping, Wolska-Kuśnierz, Pac, Tkazcyk, Haerynck, Akar, Formánková, Freiberger, Svatoň, Šedivá, Arriba-Méndez, Orfao, van Dongen and van der Burg.)

Background: Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown., Objective: We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses., Methods: Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs., Results: Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA + PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA + PCs with mild versus severe smIgA + MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA + and smIgG + MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27 + MBCs with almost normal IgG 3 + MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG 2 + MBCs; and (6) with IgA 1 + MBCs., Conclusion: Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the fact that 80% of adult acute myeloid leukaemia patients reach complete morphological remission after induction chemotherapy, many of them relapse. Many studies have shown that detection of minimal residual disease (defined as 'any detectable evidence of persistent leukaemic cells during complete morphological remission') has an added value in prediction of relapse and survival, and is more than just a surrogate marker for already known risk factors in AML. As such, the behaviour of the disease during treatment might become equally or even more important to decide whether or not an upgrade of treatment (such as an allogeneic stem cell transplantation) is necessary to improve outcome. However, there are still many open issues as to what the ideal time point is to measure MRD, which threshold is clinically significant, what sample (peripheral blood or bone marrow) should be used and how we can standardize tests so that results from different labs become comparable. This review gives an overview of currently available evidence regarding technical issues, prognostic impact and MRD-directed treatment in AML., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Cancer-related gene expression data mostly originate from unfractionated bulk samples, leading to "expression averaging" of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single-cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM-based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels., Methods: Neuroblastoma (NB) cell lines were studied for MYCN gene expression by PrimeFlow™ and compared with the gold standard, RT-qPCR. Dilution series of NB cells (0.10-11%) were prepared to evaluate performance in small cell populations. Diagnostic material of de novo acute myeloid leukemia (AML) patients was used to measure Wilms' tumor 1 (WT1) expression in bulk leukemic cells and rare subsets, e.g. leukemic stem cells (LSCs). FCM analysis was performed on a FACSCanto II (BD Biosciences) using Infinicyt™ (Cytognos ® ) for data analysis. mRNA expression was reported by normalized mean fluorescence intensity (MFI) values and staining indices., Results: MYCN mRNA quantified by PrimeFlow™ significantly correlated with RT-qPCR and remained detectable in small (0.1%) populations. Using PrimeFlow TM , WT1 levels were shown to be significantly higher in AML patient samples with WT1 overexpression, previously defined by RT-qPCR. Moreover, WT1 overexpression was distinguishable between heterogeneous cell populations and remained measurable in rare LSCs., Conclusion: PrimeFlow™ is a sensitive technique to investigate mRNA expressions, with high concordance to RT-qPCR. High (MYCN) and subtle (WT1) overexpressed mRNA targets can be quantified in heterogeneous and rare subpopulations e.g. LSCs. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Background: Light microscopy and antigen-based rapid diagnostic tests are the primary diagnostic tools for detecting malaria, although being labour-intensive and frequently challenged by lack of personnel's experience and low levels of parasite density. The latter being especially important in non-endemic settings. Novel molecular techniques aim to overcome this drawback. The objective of this study was to assess the diagnostic performance of the illumigene malaria assay ® (Meridian Bioscience) compared to microscopy, RDT and real-time PCR. This loop-mediated isothermal amplification (LAMP) assay is a qualitative in vitro diagnostic test for the direct detection of Plasmodium spp. DNA in human venous whole blood samples., Methods: The illumigene assay was assessed on a retrospective panel of stored blood samples (n = 103) from returned travellers and external quality control samples (n = 12). Additionally the assay was prospectively assessed on 30 fresh routine samples with a request for malaria diagnosis. The illumigene assay was compared to microscopy, RDT and Plasmodium species specific real-time PCR., Results: In the retrospective evaluation, the illumigene assay showed 100% agreement with the real-time PCR, RDT and microscopy yielding a sensitivity and specificity of 100% (95% CI 95.1-100% and 89.7-100%, respectively). Seven samples from patients recently treated for Plasmodium falciparum infection that were RDT positive and microscopy negative yielded positive test results. The performance of the illumigene assay equals that of microscopy combined with RDT in the prospective panel with three false negative RDT results and one false negative microscopy result. Excellent concordance with PCR was observed. The limit of detection of the assay approached 0.5 parasites/µL for both P. falciparum and Plasmodium vivax., Conclusion: In non-endemic regions where the diagnostic process for malaria infections is questioned by lack of experience and low levels of parasite densities, the illumigene assay can be of value. Due to its high sensitivity, the LAMP assay may be considered as primary diagnostic test. The results of this study indicate that negative screen results do not need further confirmation. However, before implementation, this approach needs to be confirmed in larger, prospective studies. A shortcoming of this assay is that no species identification nor determination of parasite density are possible.

The etiology of primary antibody deficiencies is largely unknown. Beside rare monogenic forms, the majority of cases seem to have a more complex genetic basis. Whereas common variable immunodeficiency has been investigated in depth, there are only a few reports on milder primary antibody deficiencies such as idiopathic primary hypogammaglobulinemia and IgG subclass deficiency. We performed flow cytometric immunophenotyping in 33 patients with common variable immunodeficiency, 23 with idiopathic primary hypogammaglobulinemia and 21 with IgG subclass deficiency, as well as in 47 asymptomatic first-degree family members of patients and 101 unrelated healthy controls. All three groups of patients showed decreased memory B- and naïve T-cell subsets and decreased B-cell activating factor receptor expression. In contrast, circulating follicular helper T-cell frequency and expression of inducible T-cell co-stimulator and chemokine receptors were only significantly altered in patients with common variable immunodeficiency. Asymptomatic first-degree family members of patients demonstrated similar, albeit intermediate, alterations in naïve and memory B- and T-cell subsets. About 13% of asymptomatic relatives had an abnormal peripheral B-cell composition. Furthermore, asymptomatic relatives showed decreased levels of CD4 + recent thymic emigrants and increased central memory T cells. Serum IgG and IgM levels were also significantly lower in asymptomatic relatives than in healthy controls. We conclude that, in our cohort, the immunophenotypic landscape of primary antibody deficiencies comprises a spectrum, in which some alterations are shared between all primary antibody deficiencies whereas others are only associated with common variable immunodeficiency. Importantly, asymptomatic first-degree family members of patients were found to have an intermediate phenotype for peripheral B- and T-cell subsets., (Copyright© Ferrata Storti Foundation.)

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive stem cell disease of early childhood. RAS activation constitutes the core component of oncogenic signaling. In addition, leukemic blasts in one-fourth of JMML patients present with monosomy 7, and more than half of patients show elevated age-adjusted fetal hemoglobin (HbF) levels. Hematopoietic stem cell transplantation is the current standard of care and results in an event-free survival rate of 50% to 60%, indicating that novel molecular-driven therapeutic options are urgently needed. Using gene expression profiling in a series of 82 patient samples, we aimed at understanding the molecular biology behind JMML and identified a previously unrecognized molecular subgroup characterized by high LIN28B expression. LIN28B overexpression was significantly correlated with higher HbF levels, whereas patients with monosomy 7 seldom showed enhanced LIN28B expression. This finding gives a biological explanation of why patients with monosomy 7 are rarely diagnosed with high age-adjusted HbF levels. In addition, this new fetal-like JMML subgroup presented with reduced levels of most members of the let-7 microRNA family and showed characteristic overexpression of genes involved in fetal hematopoiesis and stem cell self-renewal. Lastly, high LIN28B expression was associated with poor clinical outcome in our JMML patient series but was not independent from other prognostic factors such as age and age-adjusted HbF levels. In conclusion, we identified elevated LIN28B expression as a hallmark of a novel fetal-like subgroup in JMML., (© 2016 by The American Society of Hematology.)

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728., (Copyright© Ferrata Storti Foundation.)

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage., (Copyright© Ferrata Storti Foundation.)

Background: Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored., Design and Methods: Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation., Results: High levels of RHAMM were expressed by CD34(+)CD38(+) and CD34(-) acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34(+)CD38(-) leukemic stem cells, and was not different from that in CD34(+)CD38(-) hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34(+) cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells., Conclusions: RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior., (Copyright © 2011 UICC.)

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup., (© 2011 Blackwell Publishing Ltd.)

Introduction: Apoptosis is one of the mechanisms behind successful chemotherapy and radiation treatment. Radiolabeled annexin A5 has been demonstrated to be a successful tool in the detection of apoptosis following chemotherapy in vivo., Methods: His-tagged annexin A5 was labeled with [(99m)Tc]-tricarbonyl and evaluated as apoptosis imaging radiotracer in vitro and in vivo. The binding of the radiotracer was evaluated in Colo205 cells stimulated with 5-FU (1 mM) for 4 and 24 h, and confirmed by flow cytometry. Biodistribution and dosimetric studies were performed in healthy nude mice (n=5) via planar scintigraphy. [(99m)Tc]-(CO)(3) His-annexin A5 was also evaluated for in vivo imaging of spontaneous apoptosis in Colo205-bearing mice (n=12)., Results: The labeling procedure yielded a compound with 95-99% radiochemical purity and good in vitro stability. In vitro binding experiments indicated that the radiotracer retained its PS-binding activity. [(99m)Tc]-(CO)(3) His-annexin A5 rapidly cleared from the blood and predominantly accumulated in the kidneys. Absorbed dose (per organ) was found to be 116 ± 64 μGy/MBq for the kidneys and 10.38 ± 0.50 μGy/MBq for the liver. The effective dose was 7.00 ± 0.28 μSv/MBq. Spontaneous apoptosis in Colo205-bearing mice was visualised by [(99m)Tc]-(CO)(3) His-annexin A5 SPECT and correlated well with caspase-3 immunostaining (R=0.867, P<.01)., Conclusion: [(99m)Tc]-(CO)(3) His-annexin A5 may be a useful novel radioligand for the in vivo detection of cell death associated with PS expression. A simple, noninvasive way of detecting apoptosis in vivo could have many applications including a better understanding of the extent and timing of apoptosis in response to cancer therapies and assessment of early tumor response., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK&#95;blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Background: We report a case of temporary impaired hemoglobin scavenging in a patient with an acute HIV-1 retroviral syndrome. The patient was presented at the emergency department in a severe inflammatory state, mimicking bacterial sepsis and/or hemophagocytic syndrome. The serum showed a hemolytic aspect. In contrast, serum haptoglobin concentration was not decreased., Methods: The hemolysis index was determined and the visual absorbance spectroscopy spectrum of the serum was studied. alpha1 microglobulin and hemopexin concentrations were determined in serum. The presence of circulating hemoglobin:haptoglobin complexes in serum and the saturation of the haptoglobin were investigated using starch gel electrophoresis followed by peroxidase staining. CD163 expression on peripheral blood monocytes was analyzed using flow cytometry., Results: A temporarily impaired hemoglobin scavenging was documented by an increased hemolysis index, absence of decreased haptoglobin levels, presence of circulating hemoglobin:haptoglobin complexes in serum and decreased hemopexin and alpha1 microglobulin concentrations., Conclusions: A temporarily impaired hemoglobin scavenging was observed due to a transient CD163 pathway impairment following an acute HIV-1 retroviral syndrome. The patient improved clinically and biochemically after initiation of HIV-1 anti-retroviral therapy. The data suggest a transient HIV-1 mediated CD163 impairment, although a latent drug mediated block could not be ruled out completely., (2010 Elsevier B.V. All rights reserved.)

Background: Chronic lymphocytic leukemia (CLL) is an incurable disease with a natural history of increasing resistance to chemotherapy. A novel approach to overcome chemotherapy resistance may be targeting the endoplasmic reticulum (ER)., Patients and Methods: The involvement of the unfolded protein response (UPR) in the cell killing effect of xanthohumol (X) was examined in 18 patient samples., Results: X-induced apoptosis of CLL cells was accompanied by the induction of glucose-regulated protein of 78 kDa (GRP78) and heat-shock protein of 70 kDa (Hsp70) protein levels and by sustained phosphorylation of the eukaryotic translation initiation factor 2 (eIF2alpha), suggesting the involvement of the ER stress transducer, the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK). The X-box-binding protein 1 (XBP1) mRNA was spliced but no clear activation of activating transcription factor 6 (ATF6) was observed. The proapoptotic outcome was further demonstrated by the up-regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), down-regulation of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), cleavage of poly-(ADP)-ribose polymerase (PARP) and processing of caspase-3, -4 and -9. Furthermore, X showed proteasome inhibitory activity., Conclusion: X stimulates the proapoptotic arm of the UPR in ex vivo CLL cells, suggesting that ER stress may play an important role during X-induced apoptosis.

Hop (Humulus lupulus L.) is an essential ingredient of beer, where it provides the typical bitter taste, but is also applied in traditional folk medicine for sedative and antibacterial purposes. In this study, we demonstrate and compare the anti-inflammatory effect of various classes of hop bitter acids (HBA), including alpha-acids (AA), beta-acids (BA), and iso-alpha-acids (IAA), in fibroblasts, which are important players in the inflammatory response. All three studied classes of HBA blocked the tumor necrosis factor alpha (TNF)-induced production of the cytokine IL6, and inhibited the transactivation of the pro-inflammatory transcription factors nuclear factor kappa B (NF-kappaB), activator protein-1 (AP-1), and cAMP-response element-binding protein (CREB). In this respect, the six-membered ring compounds AA and BA showed equal potency, whereas the five-membered ring compounds, IAA, were effective only when used at higher concentrations. Furthermore, with regard to the mechanism of NF-kappaB suppression, we excluded a possible role for glucocorticoid receptor alpha (GRalpha), peroxisome proliferators-activated receptor alpha/gamma (PPARalpha or PPARgamma), nuclear receptors (NRs) that are also known to inhibit inflammation by directly interfering with the activity of pro-inflammatory transcription factors. Interestingly, combining hop acids and selective agonists for GRalpha, PPARalpha, or PPARgamma resulted in additive inhibition of NF-kappaB activity after TNF treatment, which may open up new avenues for combinatorial anti-inflammatory strategies with fewer side effects. Finally, systemic administration of HBA efficiently inhibited acute local inflammation in vivo.

Summary: The clinical course of individual CLL patients is highly variable, with life expectancies ranging from months to decades. Importantly, a significant subset of patients presents with low grade CLL, but will nevertheless develop a more aggressive and life-threatening disease. As these patients may potentially benefit from early treatment, it is crucial to assess patients' prognosis at diagnosis, allowing individual risk-adapted therapy. Reliable predictions of prognosis in an early stage of the disease have long been lacking in the clinical workup of CLL patients. During the last decades many efforts have been made to identify prognostic markers in CLL, resulting in a plethora of reports describing the predictive value of different parameters with regard to overall survival, disease progression and response to therapy. In this review, we attempt to provide an overview of the literature and we discuss the most important prognostic markers in CLL, from clinical staging systems and serum markers over proliferation markers and cytogenetics to more recent markers like the IgV(H) mutation status and its possible surrogate markers. Particular attention is paid to the advantages and drawbacks of all different markers, both from a clinical and from a technical point-of-view, highlighting the accomplishments as well as the remaining challenges in this rapidly evolving area of CLL research. Although the great majority of prognostic markers is not included in current international treatment guidelines, several of these markers deserve to be evaluated in prospective clinical trials and may eventually contribute to an improved clinical management of CLL patients.

Introduction: Heparin-induced thrombocytopenia (HIT) remains a very challenging diagnosis. The first objective of this study was to compare the performance of the ID-H/PF4 PaGIA with the Asserachrom HPIA ELISA. The main purpose was to evaluate the diagnostic utility of the combination of the H/PF4 PaGIA with the clinical "4T's" score as a screening strategy., Materials and Methods: 102 patients with clinical suspicion of HIT were classified into risk groups using the 4T's score. The presence of HIT antibodies was assessed by two immunoassays and confirmed by a functional flow cytometric assay., Results: Comparison of the ID-H/PF4 PaGIA with the Asserachrom HPIA ELISA demonstrated a comparable technical performance, being an excellent screening test to rule out HIT (negative predictive value or NPV=100%). According to the 4T's score, HIT was excluded in all low risk patients (NPV=100%). ELISA optical density levels were significantly different between all risk groups (P-values<0.01). In contrast, due to the low positive predictive value (22%) and weak positive likelihood ratio (2.6), a positive ID-H/PF4 PaGIA result did not considerably increase the probability of HIT., Conclusion: Our study confirms the combination of the 4T's score with the ID-H/PF4 PaGIA as a reliable strategy to rule out HIT. Yet, confirming positive ID-H/PF4 PaGIA results by flow cytometry within 1-2 h after blood sampling remains necessary. This novel clinical-laboratory approach can contribute in a rapid and reliable way to the definite diagnosis of HIT.

ABO-incompatible (ABO-I) liver transplantation is a controversial issue because of the generally less favorable outcome as compared to compatible transplants. Encouraging results have been shown by the introduction of new strategies to reduce posttransplant-specific hemagglutinin (HA) titers with plasmapheresis, reinforced immunosuppression (IS), and the use of splenectomy. We describe a new protocol consisting of daclizumab (DAC) induction, mycophenolate mofetil (MMF)/tacrolimus (TAC)/steroids without splenectomy. Five recipients (mean age of 47 +/- 14 yr) undergoing ABO-I living donor liver transplantation (LDLT) were included in this protocol. Immunoadsorbent columns (Glycosorb ABO) were used for antigen-specific immunoadsorption (ASI). The median follow-up was 18.5 +/- 10.5 months. ASI was very efficient in lowering HA titers (mean log(2) immunoglobulin [Ig] M [IgM] and IgG values before and after ASI were 5.9 +/- 2.8 and 1.2 +/- 1.4 [P= 0.0038] and 6.5 +/- 2.3 and 1.1+/- 1.9, respectively [P= 0.0001]). Persisting low HA titers were observed over time. No sepsis nor cytomegalovirus infection episodes were recorded. Acute cellular rejection (ACR) occurred in 1 recipient responding to steroid pulse therapy. Two grafts were lost in 2 patients due to technical failure during the first postoperative month. We conclude that ASI using Glycosorb ABO, quadruple immunosuppression including DAC and MMF provide high efficiency to lower HA titers over time, avoiding the need for splenectomy. ABO-I LDLT can be performed with this adapted IS protocol.

Background: External beam radiotherapy and beta-radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha-emitters is attractive, owing to the high (LET) nature and short path length of alpha particles allowing for higher tumor cell kill and lower toxicity to healthy tissues., Objectives: The aim of this study was to assess the response of B-Cell chronic lymphocytic leukemia (B-CLL) cells in vitro after treatment with chemotherapy (cisplatin, fludarabine, doxorubicin, or vincristine) or other pharmaceuticals (colchicine, simvastatin, or cyclosporin A) in combination with (60)Co-gamma or (213)Bi-alpha-irradiation., Methods: (213)Bi was eluted from a (225)Ac generator. Apoptosis was scored by flow cytometric analysis of the cells stained with Annexin-V and 7 amino actinomycin D. Metabolic activity was assessed by a MTT assay., Results: The response induced by alpha- irradiation is systematically higher than the response induced by gamma-irradiation. The combination of drug treatment with alpha-irradiation induced a systematic, higher response, compared to treatment with drugs alone, even for the highest concentrations used. For all the drugs used in this study, synergism or additivity was demonstrated for the combination of drugs and radiotherapy with a stronger effect for alpha-particles., Conclusions: The results of this in vitro study highlight a potential benefit of alpha-irradiation in combination with the drugs considered in this study.

Background: Several studies have shown that longitudinal systolic function and left ventricular filling pressures, as assessed with tissue Doppler imaging, predict exercise capacity., Aim: The aim of this study was to evaluate whether natriuretic peptides and inflammatory parameters can independently predict maximum oxygen uptake at peak exercise (VO2max) on top of tissue Doppler imaging-derived markers., Methods: We evaluated 142 patients (age 70 +/- 6 years, 77% men) with known or suspected coronary artery disease and a preserved left ventricular ejection fraction (> or = 50%). All patients underwent bicycle spiroergometry, and N-terminal pro-B-type natriuretic peptide levels were determined. Cytokines (IL-6 and soluble tumor necrosis factor receptors 1 and 2) and high-sensitivity C-reactive protein were measured as inflammatory markers. Tissue Doppler imaging was applied to evaluate peak long axis systolic velocities (Sm) and early mitral annulus velocities (E'). Ratio of early transmitral flow (E) to E' was assessed as marker of left ventricular filling. Analysis of variance, comparing VO2max quartiles, was used to determine univariate predictors and linear regression to determine multivariate VO2max predictors., Results: Average VO2max was 18.5 +/- 5.7 mL/kg per minute (range 6-36.6). Compared with the highest quartile, patients with low VO2max were more frequently women (P < .0001). N-terminal pro-B-type natriuretic peptide and cytokine levels were significantly higher in the lower VO2max categories. Longitudinal myocardial velocities increased, and E/E' decreased along with increasing VO2max. In multivariate linear regression analysis, VO2max was independently predicted by sex, glucose, Sm, E/E', and cytokine levels., Conclusion: Maximum oxygen uptake at peak exercise in patients with known or suspected coronary artery disease and preserved systolic function was independently predicted by inflammatory makers on top of tissue Doppler-derived systolic and diastolic function.

Background and Objectives: It is often difficult to obtain good karyotypes of cells from children with acute lymphoblastic leukemia (ALL) because of poor morphology and spreading. Detailed karyotyping can be further hampered by the presence of multiple rearrangements. Our objective was to search for cryptic rearrangements in childhood ALL., Design and Methods: A series of eight cases of childhood ALL with at least two structural defects were selected and studied by multiple color fluorescent in situ hybridization (M-FISH)., Results: Four previously not reported translocations were detected: a t(14;20) (q32;q11.2) in a 3-year old girl with T-ALL, a cryptic t(7;11)(q35;q24) in association with a t(1;14)(p32;q32) in a patient with T-ALL and two translocations possibly involving the same 6q26 region on the distal end of the long arm of chromosome 6. Further FISH analysis on the t(7;11) indicated rearrangement of the TCRB locus at 7q35 suggesting that this t(7;11) leads to overexpression of an as yet unidentified gene at 11q24. This observation also triggered further screening for TCRB rearrangements in T-ALL. FISH analysis of the t(14;20) with an IGH locus-specific probe provided evidence for an unusual rearrangement of the IGH gene, in the variable gene segment region. Finally, we also observed cryptic insertions of AF4 and ETV6 in combination with complex rearrangements, leading to MLL/AF4 and ETV6/RUNX1 gene fusions., Interpretation and Conclusions: This study underscores the importance and power of M-FISH analysis in unraveling complex karyotypes and identifying cryptic chromosomal rearrangements. It also sheds some light on the implication of cryptic TCRB rearrangements in T-ALL.

Background: External beam radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues., Aim: To assess the binding of rituximab to samples of B cell chronic lymphocytic leukaemia (B-CLL) and splenic lymphoma with villous lymphocytes (SLVL), and to evaluate the induction of apoptosis by conventional therapies as well as with Bi conjugated to rituximab., Method: 213Bi was eluted from a 225Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A''-DTPA as chelator. Binding assays with 213Bi-rituximab were correlated to antibody binding capacity obtained by flow cytometry. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-amino-actinomycin D., Results: Binding of 213Bi-rituximab was significantly lower for B-CLL compared to SLVL samples (12+/-3 and 42+/-10 213Bi atoms per cell, respectively, at 370 kBq.ml(-1)). The induction of apoptosis did not differ significantly between the two groups (B-CLL and SLVL) after external gamma irradiation or treatment with methylprednisolone and fludarabine (17+/-12% and 18+/-11%; 23+/-14% and 21+/-12%; 9+/-9% and 11+/-8%, respectively; all results expressed as percentages of all cells). Rituximab conjugated or not to 213Bi induced significantly more apoptosis in SLVL (42+/-19% and 42+/-17%) compared to B-CLL samples (27+/-12% and 6+/-8%)., Conclusion: Binding assays confirm that SLVL samples present more CD20 antigens compared to B-CLL samples. Conventional therapies such as fludarabine, methylprednisolone or external gamma irradiation induce similar responses in the two populations but SLVL samples present higher sensitivity towards 213Bi-rituximab. These data are in favour of alpha-RIT in SLVL patients.

External source radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. We assessed the relative biological efficacy (RBE) of alpha RIT (in vitro) compared to external gamma irradiation with respect to induction of apoptosis in B chronic lymphocytic leukaemia (B-CLL) and induction of chromosomal damage in healthy donor B and T lymphocytes. The latter was measured by a micronucleus assay. 213Bi was eluted from a 225Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A"-DTPA as a chelator. B-CLL cells from five patients were cultured for 24 h in RPMI/10% FCS while exposed to 213Bi conjugated to CD20 antibody or after external 60Co gamma irradiation. Binding assays were performed in samples of all patients to calculate the total absorbed dose. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-AAD. Apoptosis was expressed as % excess over spontaneous apoptosis in control. Full dose range experiments demonstrated 213Bi-conjugated CD20 antibody to be more effective than equivalent doses of external gamma irradiation, but showed that similar plateau values were reached at 10 Gy. The RBE for induction of apoptosis in B-CLL was 2 between 1.5 and 7 Gy. The micronucleus yield in lymphocytes of healthy volunteers was measured to assess the late toxicity caused by induction of chromosomal instability. While gamma radiation induced a steady increase in micronucleus yields in B and T cells, the damage induced by 213Bi was more dramatic, with RBE ranging from 5 to 2 between 0.1 Gy and 2 Gy respectively. In contrast to gamma irradiation, 213Bi inhibited mitogen-stimulated mitosis almost completely at 2 Gy. In conclusion, high-LET targeted alpha particle exposure killed B-CLL cells more effectively than did external gamma irradiation at a low dose (RBE=2), while a plateau was reached at a high dose. Long-term toxicity on healthy B and T lymphocytes was systematically higher for the alpha emitter (RBE=5 to 2).

Purpose: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion., Methods: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay., Results: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent., Conclusions: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.

Objective: To assess the evolution of in vitro T cell response to hepatitis C virus (HCV) Core, E1, E2 and NS3 antigens in 10 patients with chronic hepatitis C, before, during and after a high dose interferon alpha (IFN-alpha) therapy, and to evaluate the influence of IFN-alpha on the in vivo and in vitro production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)., Methods: T cell response to HCV antigens was evaluated by lymphoproliferation assays. In vivo and in vitro cytokine production was evaluated at weeks 0, 4, 8, and 12 of IFN-alpha therapy by enzyme immunoassays., Results: In general, of all HCV antigens tested throughout the follow-up, those belonging to the Core region were the most immunostiumlatory. This observation was valid in IFN-alpha responders as well as IFN-alpha non-responders. The lymphoproliferative response to HCV antigens increased during IFN-alpha therapy. Serum levels of TNF-alpha were significantly increased in HCV patients, and six out of ten patients showed increased IFN-gamma serum levels. A significant decrease of IFN-gamma levels was observed during therapy and the same trend was seen for TNF-alpha. Mitogen-stimulated TNF-alpha and IFN-gamma production before therapy did not differ from that of normal controls, however, the cytokine production was reduced at week 4 of therapy, corresponding with a clinical improvement. A return to pretreatment values was observed after 8 weeks of therapy., Conclusions: a) Core antigens are the most immunostimulatory HCV antigens at the T cell level in chronic hepatitis C patients; b) High dose IFN-alpha therapy induces an increase in lymphoproliferative response to HCV antigens; c) Serum levels of TNF-alpha are increased in HCV patients; d) High dose IFN-alpha therapy induces a decrease in serum levels of IFN-gamma; e) High dose IFN-alpha therapy induces a transiently decreased mitogen-induced TNF-alpha and IFN-gamma production.