Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell–Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199–314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5–88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens

Routine use of ancillary investigations in staging diffuse large B-cell lymphoma improves the International Prognostic Index (IPI)

<p>Abstract</p> <p>Background</p> <p>The International Prognostic Index (IPI) is used to determine prognosis in diffuse large B-cell lymphoma (DLBCL). One of the determinants of IPI is the stage of disease with bone marrow involvement being classified as stage IV. For the IPI, involvement on bone marrow is traditionally defined on the basis of histology with ancillary investigations used only in difficult cases to aid histological diagnosis. This study aimed to determine the effect of the routine use of flow cytometry, immunohistochemistry and molecular studies in bone marrow staging upon the IPI.</p> <p>Results</p> <p>Bone marrow trephines of 156 histologically proven DLBCL cases at initial diagnosis were assessed on routine histology, and immunohistochemistry using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a) and kappa and lambda light chains. Raw flow cytometry data on all samples were reanalysed and reinterpreted blindly. DNA extracted from archived paraffin-embedded trephine biopsy samples was used for immunoglobulin heavy chain and light chain gene rearrangement analysis. Using immunophenotyping (flow cytometry and immunohistochemistry), 30 (19.2%) cases were upstaged to stage IV. A further 8 (5.1%) cases were upstaged using molecular studies. A change in IPI was noted in 18 cases (11.5%) on immunophenotyping alone, and 22 (14.1%) cases on immunophenotyping and molecular testing. Comparison of two revised IPI models, 1) using immunophenotyping alone, and 2) using immunophenotyping with molecular studies, was performed with baseline IPI using a Cox regression model. It showed that the revised IPI model using immunophenotyping provides the best differentiation between the IPI categories.</p> <p>Conclusion</p> <p>Improved bone marrow staging using flow cytometry and immunohistochemistry improves the predictive value of the IPI in patients with DLBCL and should be performed routinely in all cases.</p

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by diff

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1(IS) and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.</p

Development and Applications of a Yeast-based Bioassay for the Mycotoxin Zearalenone

Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic species of Fusarium. As a consequence of infection with F. culmorum and F. graminearum, ZON can be found in cereals and derived food products. Several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We have developed a sensitive yeast bioassay allowing detection of the estrogenic activity of ZON in cereal extracts without requiring further clean up steps. The high sensitivity makes this assay suitable for low cost monitoring of contamination of small grain cereals with estrogenic Fusarium mycotoxins, but also attractive as a tool for basic research. We have successfully used yeast indicator strains to screen for mutants of F. graminearum which no longer produce detectable amounts of ZON, and have identified a plant cDNA encoding a ZON detoxification enzyme

Effect of ultramarathon cycling on the heart rate in elite cyclists

Objectives: To analyse the heart rate (HR) response and estimate the ultraendurance threshold—the optimum maintainable exercise intensity of ultraendurance cycling—in ultraendurance elite cyclists competing in the Race across the Alps. Methods: HR monitoring was performed in 10 male elite cyclists during the first Race across the Alps in 2001 (distance: 525 km; cumulative altitude difference: 12 600 m) to investigate the exercise intensity of a cycle ultramarathon and the cardiopulmonary strains involved. Four different exercise intensities were defined as percentages of maximal HR (HR(max)) as follows: recovery HR (HR(re)), <70% of HR(max); moderate aerobic HR (HR(ma)), 70–80%; intense aerobic HR (HR(ia)), 80–90%; and high intensity HR (HR(hi)), >90%. Results: All athletes investigated finished the competition. The mean racing time was 27 hours and 25 minutes, and the average speed was 18.6 km/h. The mean HR(max) was 186 beats/min, and the average value of measured HRs (HR(average)) was 126 beats/min resulting in a mean HR(average)/HR(max) ratio of 0.68, which probably corresponds to the ultraendurance threshold. The athletes spent 53% (14 hours 32 minutes) of total race time within HR(re), 25% (6 hours 51 minutes) within HR(ma), 19% (5 hours 13 minutes) within HR(ia), and only 3% (49 minutes) within HR(hi), which shows the exercise intensity to be predominantly moderate (HR(re) + HR(ma) = 78% or 21 hours 23 minutes). The HR response was influenced by the course profile as well as the duration. In all subjects, exercise intensity declined significantly during the race, as indicated by a decrease in HR(average)/HR(max) of 23% from 0.86 at the start to 0.66 at the end. Conclusions: A substantial decrease (10% every 10 hours) in the HR response is a general cardiovascular feature of ultramarathon cycling, suggesting that the ultraendurance threshold lies at about 70% of HR(max) in elite ultramarathon cyclists