Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients: immunobiological characteristics and clinical implications.

DH-JH rearrangements of the Ig heavy-chain gene (IGH) occur early during B-cell development. Consequently, they are detected in precursor-B-cell acute lymphoblastic leukemias both at diagnosis and relapse. Incomplete DJH rearrangements have also been occasionally reported in mature B-cell lymphoproliferative disorders, but their frequency and immunobiological characteristics have not been studied in detail. We have investigated the frequency and characteristics of incomplete DJH as well as complete VDJH rearrangements in a series of 84 untreated multiple myeloma (MM) patients. The overall detection rate of clonality by amplifying VDJH and DJH rearrangements using family-specific primers was 94%. Interestingly, we found a high frequency (60%) of DJH rearrangements in this group. As expected from an immunological point of view, the vast majority of DJH rearrangements (88%) were unmutated. To the best of our knowledge, this is the first systematic study describing the incidence of incomplete DJH rearrangements in a series of unselected MM patients. These results strongly support the use of DJH rearrangements as PCR targets for clonality studies and, particularly, for quantification of minimal residual disease by real-time quantitative PCR using consensus JH probes in MM patients. The finding of hypermutation in a small proportion of incomplete DJH rearrangements (six out of 50) suggests important biological implications concerning the process of somatic hypermutation. Moreover, our data offer a new insight in the regulatory development model of IGH rearrangements.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

In the present paper, we report on the use of the heteroduplex PCR technique to detect the presence of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) in the apheresis products of patients with multiple myeloma (MM) undergoing autologous peripheral blood stem cell (APBSC) transplantation. Twenty-three out of 31 MM patients undergoing APBSC transplantation with VDJH segments clonally rearranged detected at diagnosis were included in the study. Samples of the apheresis products were PCR amplified using JH and VH (FRIII and FRII) consensus primers and subsequently analyzed with the heteroduplex technique, and compared with those obtained at diagnosis. 52% of cases yielded positive results (presence of clonally rearranged VDJH segments in at least one apheresis). The presence of positive results in the apheresis products was not related to any pretransplant characteristics with the exception of response status at transplant. Thus, while no one patient with positive apheresis products was in complete remission (CR), negative immunofixation, before the transplant, five cases (46%) with negative apheresis were already in CR at transplant (P = 0.01). The remaining six cases with heteroduplex PCR negative apheresis were in partial remission before transplant. Patients with clonally free products were more likely to obtain CR following transplant (64% vs 17%, P= 0.02) and a longer progression-free survival, (40 months in patients transplanted with polyclonal products vs 20 with monoclonal ones, P = 0.03). These results were consistent when the overall survival was considered, since it was better in those patients with negative apheresis than it was in those with positive (83% vs 36% at 5 years from diagnosis, P= 0.01). These findings indicate that the presence of clonality rearranged VDJH segments is related to the response and outcome in MM transplanted patients.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12 mM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50 mM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50 mM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24 h of exposure to 50 mM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10 nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins

Construção e produção de fragmentos de anticorpos anti-ssDNA em um contexto de desenvolvimento de linfócitos B

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.

Isolamento e caracterização biológica e bioquímica de um activador do factor x da coagulação presente no veneno da serpente Bothrops erythromelas

Dissertação de Mestrado Integrado em Medicina Veterinária

Étude des voies de signalisation activées par la prostaglandine F2α dans les cellules de cancer colorectal

Le cancer colorectal représente la troisième forme de cancer la plus fréquente au Canada. Malgré les récents développements, aucun traitement curatif n’est disponible pour les patients diagnostiqués à un stade avancé de la maladie. Plusieurs évidences supportent un rôle important des prostaglandines dans cette maladie. En effet, une surexpression des récepteurs de type EP et FP est remarquée. Ces derniers ainsi que les molécules de signalisations intracellulaires qu’ils activent représentent donc de nouvelles cibles pour traiter ce cancer. Nous et d’autres groupes avons démontré que les protéines G monomériques sont des petits interrupteurs moléculaires importants dans la signalisation intracellulaire engagée par les récepteurs dans des cellules saines mais qu’un dérèglement de celles-ci est associé au cancer. L’objectif principal de ce projet de recherche vise donc à identifier les voies de signalisations par lesquelles le récepteur FP contribue aux capacités invasives des cellules tumorales d’origine colorectale. Notre hypothèse est que la protéine ARF6, une des 6 isoformes des ARFs, et la protéine RhoA, agissent pour coordonner l’activation des voies de signalisations associées à la migration et l’invasion cellulaire. Nos résultats ont indiqué que la stimulation des cellules HEK293 exprimant de façon stable le récepteur FP (HA-FP) ainsi que les cellules SW480, une lignée invasive de cancer colorectal, par le PGF2α augmentait les niveaux d’activation d’ARF6 mais également de la protéine RhoA. De plus, d’autres médiateurs et intermédiaires associés à la réorganisation du cytosquelette comme la cofiline et la chaine légère de la myosine (MLC) ont été hautement phosphorylés suite à la stimulation par la prostaglandine. Ces observations sont associées avec une augmentation des fibres de stress dans les cellules. Nous avons tenté de déterminer si l’inhibition de ces protéines G affectait la capacité du PGF2α à activer ces intermédiaires de signalisation ainsi que certains effets biologiques. Ainsi, nos expériences contribueront à identifier de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer colorectal.

Étude des voies de signalisation activées par la prostaglandine F2α dans les cellules de cancer colorectal

Le cancer colorectal représente la troisième forme de cancer la plus fréquente au Canada. Malgré les récents développements, aucun traitement curatif n’est disponible pour les patients diagnostiqués à un stade avancé de la maladie. Plusieurs évidences supportent un rôle important des prostaglandines dans cette maladie. En effet, une surexpression des récepteurs de type EP et FP est remarquée. Ces derniers ainsi que les molécules de signalisations intracellulaires qu’ils activent représentent donc de nouvelles cibles pour traiter ce cancer. Nous et d’autres groupes avons démontré que les protéines G monomériques sont des petits interrupteurs moléculaires importants dans la signalisation intracellulaire engagée par les récepteurs dans des cellules saines mais qu’un dérèglement de celles-ci est associé au cancer. L’objectif principal de ce projet de recherche vise donc à identifier les voies de signalisations par lesquelles le récepteur FP contribue aux capacités invasives des cellules tumorales d’origine colorectale. Notre hypothèse est que la protéine ARF6, une des 6 isoformes des ARFs, et la protéine RhoA, agissent pour coordonner l’activation des voies de signalisations associées à la migration et l’invasion cellulaire. Nos résultats ont indiqué que la stimulation des cellules HEK293 exprimant de façon stable le récepteur FP (HA-FP) ainsi que les cellules SW480, une lignée invasive de cancer colorectal, par le PGF2α augmentait les niveaux d’activation d’ARF6 mais également de la protéine RhoA. De plus, d’autres médiateurs et intermédiaires associés à la réorganisation du cytosquelette comme la cofiline et la chaine légère de la myosine (MLC) ont été hautement phosphorylés suite à la stimulation par la prostaglandine. Ces observations sont associées avec une augmentation des fibres de stress dans les cellules. Nous avons tenté de déterminer si l’inhibition de ces protéines G affectait la capacité du PGF2α à activer ces intermédiaires de signalisation ainsi que certains effets biologiques. Ainsi, nos expériences contribueront à identifier de nouvelles cibles thérapeutiques potentielles pour le traitement du cancer colorectal.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically “blocked” because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.

IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.