Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs

Individual variability in cardiac biomarker release after 30 min of high-intensity rowing in elite and amateur athletes

This study had two objectives: (i) to examine individual variation in the pattern of cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) release in response to high-intensity rowing exercise, and (ii) to establish whether individual heterogeneity in biomarker appearance was influenced by athletic status (elite vs. amateur). We examined cTnI and NT-proBNP in 18 elite and 14 amateur rowers before and 5 min, 1, 3, 6, 12, and 24 h after a 30-min maximal rowing test. Compared with pre-exercise levels, peak postexercise cTnI (pre: 0.014 ± 0.030 μg·L–1; peak post: 0.058 ± 0.091 μg·L–1; p = 0.000) and NT-proBNP (pre: 15 ± 11 ng·L–1; peak post: 31 ± 19 ng·L–1; p = 0.000) were elevated. Substantial individual heterogeneity in peak and time-course data was noted for cTnI. Peak cTnI exceeded the upper reference limit (URL) in 9 elite and 3 amateur rowers. No rower exceeded the URL for NT-proBNP. Elite rowers had higher baseline (0.019 ± 0.038 vs. 0.008 ± 0.015 μg·L–1; p = 0.003) and peak postexercise cTnI (0.080 ± 0.115 vs. 0.030 ± 0.029 μg·L–1; p = 0.022) than amateur rowers, but the change with exercise was similar between groups. There were no significant differences in baseline and peak postexercise NT-proBNP between groups. In summary, marked individuality in the cTnI response to a short but high-intensity rowing bout was observed. Athletic status did not seem to affect the change in cardiac biomarkers in response to high-intensity exercise

Integration of mutational signature analysis with 3D chromatin data unveils differential AID-related mutagenesis in indolent lymphomas

Activation-induced deaminase (AID) is required for somatic hypermutation in immunoglobulin genes, but also induces off-target mutations. Follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), the most frequent types of indolent B-cell tumors, are exposed to AID activity during lymphomagenesis. We designed a workflow integrating de novo mutational signatures extraction and fitting of COSMIC (Catalogue Of Somatic Mutations In Cancer) signatures, with tridimensional chromatin conformation data (Hi-C). We applied the workflow to exome sequencing data from lymphoma samples. In 33 FL and 30 CLL samples, 42% and 34% of the contextual mutations could be traced to a known AID motif. We demonstrate that both CLL and FL share mutational processes dominated by spontaneous deamination, failures in DNA repair, and AID activity. The processes had equiproportional distribution across active and nonactive chromatin compartments in CLL. In contrast, canonical AID activity and failures in DNA repair pathways in FL were significantly higher within the active chromatin compartment. Analysis of DNA repair genes revealed a higher prevalence of base excision repair gene mutations (p = 0.02) in FL than CLL. These data indicate that AID activity drives the genetic landscapes of FL and CLL. However, the final result of AID-induced mutagenesis differs between these lymphomas depending on chromatin compartmentalization and mutations in DNA repair pathways.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Peripheral IgE Repertoires of Healthy Donors Carry Moderate Mutation Loads and Do Not Overlap With Other Isotypes

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (SbIII) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and SbIII-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in SbIII-resistant lines and SbIII-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence

Tandem substitutions in somatic hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously

Tandem substitutions in somatic hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.Transplantation and immunomodulatio