Simultaneously Targeting Two Coupled Signalling Molecules in the Mesenchymal Stem Cell Support Efficiently Sensitises the Multiple Myeloma Cell Line H929 to Bortezomib.

Several studies have shown that diverse components of the bone marrow (BM) microenvironment play a central role in the progression, pathophysiology, and drug resistance in multiple myeloma (MM). In particular, the dynamic interaction between BM mesenchymal stem cells (BM-MSC) and MM cells has shown great relevance. Here we showed that inhibiting both PKC and NF-κB signalling pathways in BM-MSC reduced cell survival in the MM cell line H929 and increased its susceptibility to the proteasome inhibitor bortezomib. PKC-mediated cell survival inhibition and bortezomib susceptibility induction were better performed by the chimeric peptide HKPS than by the classical enzastaurin inhibitor, probably due to its greatest ability to inhibit cell adhesion and its increased capability to counteract the NF-κB-related signalling molecules increased by the co-cultivation of BM-MSC with H929 cells. Thus, inhibiting two coupled signalling molecules in BM-MSC was more effective in blocking the supportive cues emerging from the mesenchymal stroma. Considering that H929 cells were also directly susceptible to PKC and NF-κB inhibition, we showed that treatment of co-cultures with the HKPS peptide and BAY11-7082, followed by bortezomib, increased H929 cell death. Therefore, targeting simultaneously connected signalling elements of BM-MSC responsible for MM cells support with compounds that also have anti-MM activity can be an improved treatment strategy.

Comparative analysis of CD45 proteins in primate context: owl monkeys vs humans.

Transmembrane protein tyrosine phosphatase (PTPase) CD45 has been implicated in activating, differentiating and the development of different immune system cells. It regulates T-or B-cell activation during receptor-specific recognition by dephosphorylating tyrosine residues in protein kinase substrates. Aotus nancymaae, Aotus nigriceps, and Aotus vociferans CD45 nucleotide and deduced amino acid sequences are presented here, where we found 90-92% identity with the human counterpart in the nucleotide sequence and 83-86% in the amino acid sequence. Aotus CD45 alternative splicing isoforms include the same exons used in human CD45, producing several identical molecular weight nucleotide fragments. Most of the non-synonymous substitutions were found in the extracellular domain. The more conserved CD45 cytoplasmic portion has two intracellular phosphatase domains (D1 and D2) separated by a short spacer and some residues and motifs involved in signaling or molecular docking, intra- and intermolecular interactions and CD45 activity and activity regulation. All invariant residues and structural/functional motifs found in PTPases were totally conserved, suggesting that Aotus CD45 is a functional enzyme. Phylogenetic analysis has shown that the Aotus CD45 molecules are more related to the human homologs than to other reported vertebrate sequences and that the ancestral group of Aotus clade is A. vociferans. When Aotus species were compared, A. nigriceps and A. vociferans were the two most distant species, while A. nancymaae and A. nigriceps appeared to be a sister group. This could be relevant in deciding which Aotus species is to be used for studying particular immunological processes during lymphocyte activation or development.

Inmunoregulacion mediada por Calcitriol / Inmunoregulatory role of Calcitiol

El papel clásico endocrino del calcitrol (la,25-dihidroxivitamina D3) en la homeostasis del calcio es bien conocido. Los hallazgos relativamente recientes de la presencia del receptor de calcitrol en monocitos, linfocitos y diversas líneas celulares, y el hallazgo de la producción ectópica y localizada del calcitrol por los macrófagos, sugiere un papel importante de esta vitamina/hormona en eventos de inmunoregulación. A partir de la nueva evidencia experimental, es claro, a diferencia de los planteamientos iniciales que consideraban al calcitrol como un agente inmunosupresor, que esta hormona es capaz de estimular y de suprimir la función inmune, dependiendo de las condiciones de activación y del microambiente inmunológico. La información clínica disponible y la experimentación in vivo e in vitro apoyan un efecto inmunomodulador del calcitrol; éste debe ser considerado, por lo tanto, como uno mas de la larga y creciente lista de moduladores

The detection of lupus anticoagulant using the kaolin clotting time test: a comparative study of the different forms of analysis.

This work is a report of a comparative study between the different forms of analysis (curve, index, percentage and delta) of kaolin clotting time (KCT) for the detection of lupus anticoagulant. The reference values for all tests were defined from a study of 111 normal controls. 133 patients were studied, 106 with connective tissue disease and 27 with syphilis. LA was detected in 31 of the 133 patients studied. For KCT, the percentage and the delta showed greater sensitivity (61% and 65%, respectively) as opposed to the curve and the index (48% and 26%, respectively). We found significant statistical differences between the frequency of positivity of the index and the delta (p < 0.001) and between the index and the percentage (p < 0.005). Our findings show that if it is necessary to use KCT as a complementary test for the detection of LA, then it is more convenient to carry it out by using the percentage or the delta forms of analysis.

Role of calcium and erythrocyte cytoskeleton phosphorylation in the invasion of Plasmodium falciparum.

The role of calcium in the invasion of the human erythrocyte by the parasite Plasmodium falciparum was studied. The intraerythrocytic and intraparasitic concentrations of Ca2+ were modified using calcium-ionophore A23187 and the chelator EGTA. The Ca2+ inside the parasite appeared to be necessary for the normal completion of invasion. We determined that in recently invaded erythrocytes (2 h), the Ca2+ concentration increased about 10 times. Merozoite invasion produced a decrease in beta-spectrin phosphorylation and an increase in the phosphorylation of a protein with band 4.1 mobility. These changes were similar to those produced by an ionophore-mediated Ca2+ influx in uninfected erythrocytes. These facts support the idea that a calcium influx into erythrocytes might precede or accompany merozoite invasion, triggering a series of molecular events, including phosphorylation and dephosphorylation of cystoskeletal proteins.

Plasmodium falciparum: increased and multiple invasion during short periods of time.

We describe how to obtain an increased merozoite invasion of Plasmodium falciparum into human erythrocytes during short periods of time. Using this procedure, infected erythrocytes show multiple invasions (2-4 merozoites per erythrocyte), amplifying, several times, the effects of parasite entry into host cells. The procedure yields synchronous cultures (2-h age range) with parasitemia as high as 15%. It is possible to reach parasitemia of 50% or higher allowing for a 6-h invasion period.