CD34+CD19-CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19- (pre)-leukemic cells represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19-CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19- relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged.

Exploring High-Throughput Immunoassays for Biomarker Validation in Rheumatic Diseases in the Context of the Human Proteome Project.

Rheumatic diseases are high prevalence pathologies with different etiology and evolution and low sensitivity in clinical diagnosis. Therefore, it is necessary to develop an early diagnosis method which allows personalized treatment, depending on the specific pathology. The biology/disease initiative, at Human Proteome Project, is an integrative approach to identify relevant proteins in the human proteome associated with pathologies. A previously reported literature data mining analysis, which identified proteins related to osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PSA) was used to establish a systematic prioritization of potential biomarkers candidates for further evaluation by functional proteomics studies. The aim was to study the protein profile of serum samples from patients with rheumatic diseases such as OA, RA, and PSA. To achieve this goal, customized antibody microarrays (containing 151 antibodies targeting 121 specific proteins) were used to identify biomarkers related to early and specific diagnosis in a screening of 960 serum samples (nondepleted) (OA, n = 480; RA, n = 192; PSA, n = 288). This functional proteomics screening has allowed the determination of a panel (30 serum proteins) as potential biomarkers for these rheumatic diseases, displaying receiver operating characteristics curves with area under the curve values of 80-90%.

Genomic profiling of sporadic liver metastatic colorectal cancer.

Sporadic colorectal cancer (sCRC) is the third leading cause of cancer death in the Western world. Approximately, a quarter of sCRC patients present metastatic dissemination at the moment of diagnosis, the liver being the most frequently affected organ. Additionally, this group of CRC patients is characterized by a worse prognosis. In the last decades, significant technological developments for genome analysis have fostered the identification and characterization of genetic alterations involved in the pathogenesis of sCRC. However, genetic alterations involved in the metastatic process through which tumor cells are able to colonize other tissues with a different microenvironment, still remain to be fully identified. Here, we review current knowledge about the most relevant genomic alterations involved in the liver metastatic process of sCRC, including detailed information about the genetic profile of primary colorectal tumors vs. their paired liver metastases.

Enhanced in vitro anticancer activity of yeast expressed recombinant glucose oxidase versus commercial enzyme.

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.

ASTEMIZOLE, AN INHIBITOR OF ETHER-À-GO-GO-1 POTASSIUM CHANNEL, INCREASES THE ACTIVITY OF THE TYROSINE KINASE INHIBITOR GEFITINIB IN BREAST CANCER CELLS.

BACKGROUND: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression. OBJECTIVE: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells. MATERIALS AND METHODS: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using dose-response curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy-tometry. RESULTS: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle. CONCLUSIONS: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1.

Self-assembling functional programmable protein array for studying protein-protein interactions in malaria parasites.

BACKGROUND: Plasmodium vivax is the most widespread malarial species, causing significant morbidity worldwide. Knowledge is limited regarding the molecular mechanism of invasion due to the lack of a continuous in vitro culture system for these species. Since protein-protein and host-cell interactions play an essential role in the microorganism's invasion and replication, elucidating protein function during invasion is critical when developing more effective control methods. Nucleic acid programmable protein array (NAPPA) has thus become a suitable technology for studying protein-protein and host-protein interactions since producing proteins through the in vitro transcription/translation (IVTT) method overcomes most of the drawbacks encountered to date, such as heterologous protein production, stability and purification. RESULTS: Twenty P. vivax proteins on merozoite surface or in secretory organelles were selected and successfully cloned using gateway technology. Most constructs were displayed in the array expressed in situ, using the IVTT method. The Pv12 protein was used as bait for evaluating array functionality and co-expressed with P. vivax cDNA display in the array. It was found that Pv12 interacted with Pv41 (as previously described), as well as PvMSP142kDa, PvRBP1a, PvMSP8 and PvRAP1. CONCLUSIONS: NAPPA is a high-performance technique enabling co-expression of bait and query in situ, thereby enabling interactions to be analysed rapidly and reproducibly. It offers a fresh alternative for studying protein-protein and ligand-receptor interactions regarding a parasite which is difficult to cultivate (i.e. P. vivax).

Screening and Validation of Novel Biomarkers in Osteoarticular Pathologies by Comprehensive Combination of Protein Array Technologies.

Osteoarthritis (OA) is one of the most prevalent articular diseases. The identification of proteins closely associated with the diagnosis, progression, prognosis, and treatment response is dramatically required for this pathology. In this work, differential serum protein profiles have been identified in OA and rheumatoid arthritis (RA) by antibody arrays containing 151 antibodies against 121 antigens in a cohort of 36 samples. Then the identified differential serum protein profiles have been validated in a larger cohort of 282 samples. The overall immunoreactivity is higher in the pathological situations in comparison with the controls. Several proteins have been identified as biomarker candidates for OA and RA. Most of these biomarker candidates are proteins related to inflammatory response, lipid metabolism, or bone and extracellular matrix formation, degradation, or remodeling.

Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca2+ concentrations ([Ca2+]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca2+ oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 µg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization.

Uterine flushings from women treated with levonorgestrel affect sperm functionality in vitro.

Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH + 1 of a control cycle (CTR-LH + 1) and after receiving LNG (LNG-LH + 1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH + 1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum.

GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP(+) cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP(+) cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 µM for GABA. The evoked currents were inhibited by bicuculline (100 µM) and TPMPA (IC50, 5.9 ± 0.6 µM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein-protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A-GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP(+) cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 µm and a net distance traveled of 1-2 µm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP(+) cells during early postnatal development.