Ibrutinib serves as an efficient second-line therapy in relapsed/refractory mantle cell lymphoma. However, resistance to the BTK inhibitor results in a poor prognosis for patients. Since the mechanisms leading to resistance in initially responding tumor cells are poorly understood, this work aimed to decipher acquired features in ibrutinib-surviving cells of a sensitive mantle cell lymphoma cell line and evaluate these potential therapeutic targets in ibrutinib-treated mantle cell lymphoma. Time-resolved single-cell RNA sequencing was performed to track the transcriptomic evolution of REC-1 cells across 6 and 48 hours of treatment. Single-cell analysis uncovered a subpopulation of REC-1 with potentially greater aggressiveness and survival advantage by benefiting from interaction with the tumor microenvironment. Upregulation of B-cell receptor genes, elevated surface antigen expression of CD52 and metabolic rewiring to higher dependence on oxidative phosphorylation were identified as further potential resistance features of ibrutinib-surviving cells. RNA sequencing after prolonged incubation corroborated the increase in CD52 and oxidative phosphorylation as dominant characteristics of the cells surviving the 4-day treatment, highlighting their potential as therapeutic targets in combination with ibrutinib treatment. Concomitant use of ibrutinib and the oxidative phosphorylation inhibitor IACS-010759 increased toxicity compared to ibrutinib monotherapy due to higher apoptosis and greater inhibition of proliferation. For anti-CD52 therapy, a consecutive approach with ibrutinib pretreatment followed by incubation of surviving cells with a CD52 monoclonal antibody and human serum yielded a synergistic effect, as ibrutinib-surviving mantle cell lymphoma cells were rapidly depleted by complement-dependent cytotoxicity. Regarding the effects on primary tumor cells from mantle cell lymphoma patients, ibrutinib induced upregulation of CD52 in some cases, and increased toxicity of anti-CD52 therapy was observed in ibrutinib-sensitive patient samples after pretreatment with the BTK inhibitor. The likely favorable in vivo efficacy of an anti-CD52 therapy might therefore be restricted to a subgroup of mantle cell lymphoma patients, also in view of the associated side effects. Given the need for new therapeutic options in mantle cell lymphoma to overcome resistance to ibrutinib, this work highlights the potentially beneficial use of an oxidative phosphorylation inhibitor as add-on therapy. In addition, the findings suggest to further assess the value of anti-CD52 therapy as consolidation to ibrutinib in ibrutinib-sensitive patients with elevated CD52 surface levels on tumor cells to target resistant clones and minimize risk of minimal residual disease and relapse. / Ibrutinib dient als wirkungsvolle Zweitlinientherapie beim rezidivierten/refraktären Mantelzell-Lymphom. Jedoch führt eine Resistenz gegen den BTK-Inhibitor zu einer schlechten Prognose für die Patienten. Da die Mechanismen, die zur Resistenz in initial ansprechenden Tumorzellen führen, nur unzureichend bekannt sind, hatte diese Arbeit zum Ziel, erworbene Eigenschaften in Ibrutinib-überlebenden Zellen einer sensitiven Mantelzell-Lymphom Zelllinie aufzudecken und darauf basierende therapeutische Ansätze im Ibrutinib-behandelten Mantelzell-Lymphom zu evaluieren. Eine Einzelzell-RNA-Sequenzierung wurde durchgeführt, um die Entwicklung des Transkriptoms der REC-1 Zellen über eine Behandlung von 6 und 48 Stunden zu verfolgen. Die Einzelzellanalyse offenbarte eine Subpopulation der REC-1 Zelllinie, die möglicherweise eine größere Aggressivität und einen Überlebensvorteil aufwies, da sie von der Interaktion mit dem Tumormikromilieu profitieren könnte. Die Hochregulation von B-Zell Rezeptor Genen, erhöhte Oberflächenantigenexpression von CD52 und eine Umstellung des Metabolismus auf eine stärkere Abhängigkeit von der oxidativen Phosphorylierung wurden als weitere mögliche Resistenzeigenschaften der Ibrutinib-überlebenden Zellen identifiziert. Eine RNA-Sequenzierung nach längerer Inkubation bestätigte den Anstieg von CD52 und oxidativer Phosphorylierung als dominante Merkmale der Zellen, die die 4-tägige Behandlung überlebten, was ihr Potential als therapeutische Ansatzpunkte in Kombination mit einer Ibrutinib Therapie hervorhob. Die gleichzeitige Anwendung von Ibrutinib und dem Inhibitor der oxidativen Phosphorylierung, IACS-010759, erhöhte die Toxizität im Vergleich zur Ibrutinib-Monotherapie aufgrund einer gesteigerten Apoptose und einer stärkeren Hemmung der Proliferation. Bei der Anti-CD52-Therapie erzielte der konsekutive Ansatz mit Ibrutinib-Vorbehandlung und anschließender Inkubation der überlebenden Zellen mit einem monoklonalen CD52-Antikörper und humanem Serum einen synergistischen Effekt, da die unter Ibrutinib überlebenden Zellen schnell durch die komplementabhängige Zytotoxizität eliminiert wurden. Hinsichtlich der Auswirkungen auf primäre Tumorzellen von Mantelzell-Lymphom-Patienten führte Ibrutinib in manchen Fällen zu einer Hochregulierung von CD52, und in Ibrutinib-empfindlichen Patientenproben wurde nach Vorbehandlung mit dem BTK-Inhibitor eine erhöhte Toxizität der Anti-CD52-Therapie beobachtet. Die wahrscheinlich günstige In-vivo-Wirkung einer Anti-CD52-Therapie könnte daher auf eine Subgruppe von Mantelzell-Lymphom Patienten beschränkt sein, auch im Hinblick auf die damit verbundenen Nebenwirkungen. Angesichts des Bedarfs an neuen therapeutischen Optionen beim Mantelzell-Lymphom zur Überwindung der Ibrutinib-Resistenz unterstreicht diese Arbeit den potentiell vorteilhaften Einsatz eines Inhibitors der oxidativen Phosphorylierung als Zusatztherapie. Darüber hinaus legen die Ergebnisse nahe, den Nutzen einer Anti-CD52-Therapie als Konsolidierung zu Ibrutinib bei Ibrutinib-empfindlichen Patienten mit erhöhter CD52 Oberflächenexpression auf Tumorzellen näher zu untersuchen, um resistente Klone zu beseitigen und das Risiko einer minimalen Resterkrankung und eines Rückfalls zu minimieren.

RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re-exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells.

This thesis investigates similarities and differences in the chemistry of the Linear B clay tablets and sealings found at the Palace of Nestor in Pylos, located in the western Peloponnese, Greece. Their chemistry provides clues regarding the flow of material goods in and out of the palace and therefore to the degree of centralization of the political-economy. Over a thousand 3,000 year-old clay tablets and sealings currently housed at the National Archaeological Museum in Athens were analyzed using a pXRF over the course of the summers of 2015 and 2016. The chemical compositions were analyzed statistically. Results of the study and the conclusions are presented here.

No description available.

Endometriosis is a steroid-dependent common gynecological condition characterized by the growth of endometrial epithelial and stromal cells outside of its cavity. Estrogen dependence, progesterone resistance, in situ estrogen production, increased inflammation, resistance to apoptosis, active cell growth and proliferation, and overall disease’s heterogeneity makes it challenging to treat patients without compromising fertility. BDNF and its high affinity receptor TrkB has been shown to be dysregulated in women with endometriosis suggesting their role in disease progression. Immunocompromised mice (Rag2γc) (n=25) underwent an implantation surgery during which a cell suspension consisting of human endometrioma cells was injected into the animals’ peritoneal cavity. Upon lesion stablishment all animals were divided into five groups and treated with either high (7.5mg/kg/day), medium (5mg/kg/day) or low (2.5mg/kg/day) dose of TrkB inhibitor cyclotraxin-b, negative control (saline) or 0.04mg/kg/day letrozole. After four weeks of treatment all animals were sacrificed, all major organs were collected and assessed with routine histology for potential adverse effects of the treatment, all endometriotic implants were analyzed with routine histology and IHC for a panel of markers: BDNF, TrkB, anti-human mitochondrial protein, CD31, VEGFa, VEGFR1, VEGFR2. Treatment did not cause any significant side effects. There was a dose-dependent trend in reduction of endometriotic implants’ volume and number. IHC confirmed expression of the angiogenic markers within endometriotic implants. A larger study may be required to replicate results and advance the search for novel non-hormonal endometriosis treatment. / Thesis / Master of Science in Medical Sciences (MSMS) / Endometriosis is a chronic pain condition affecting more than 176 million women worldwide. Despite its commonality and severity its gold standard diagnosis is performed through invasive laparoscopy and there is no cure resulting in low quality of both physical and mental health of the affected individuals. Levels of a neurotrophic factor - BDNF and its high affinity receptor TrkB have been found increased in women with endometriosis and thus, BDNF-TrkB signaling cascade appears to be a promising therapeutic target. This study investigated an effect of TrkB inhibitor – cyclotraxin-b on reduction of endometriotic lesion number and volume within an immunocompromised model. Replication of results and increase of sample size may provide more details about cyclotraxin-b clinical relevance for treatment of endometriosis.

The human ABF-1 gene is expressed in activated B-cells and Epstein-Barr virus immortalized lymphoblastoid cell lines. ABF-1 represents the only member belonging to the basic helix-loop-helix (bHLH) family of transcription factors whose expression pattern is restricted to B cells. ABF-1 forms heterodirner complexes with E2A to modulate gene transcription. Here we report the isolation often novel ABF-1 splice products from a human B-cell plasmacytoma cell line. Analysis of the nucleotide sequence revealed approximately five clones harboring changes within the coding region of the reported full-length ABF-1 eDNA. In some cases, these changes resulted in the elimination of the coding region specifying the bHLH domain and likely affect DNAbinding and dirnerization of ABF-1. For the other retrieved splice products, the changes within the eDNA were localized to the 3' untranslated region (UTR), which likely indicate the potential for translational regulation of the ABF -1 gene.

No description available.

Anthrax Vaccine Adsorbed (AVA) immunization protects against anthrax disease by eliciting a neutralizing antibody response. However, antigen-specific antibody concentrations are not observed in high quantities until three immunizations have been administered over six months. Even then, humoral responses to AVA do not provide long-term immunity without an annual booster. We followed six healthy volunteers over the five-dose, 18-month AVA schedule to characterize the genetics of the immunoglobulin repertoire during the vaccination series. Two tiers of data were collected: 1) Immunoglobulin variable region genes (IgVRG) from bulk sorted naïve, memory and plasmablast (PB) B cells and 2) single cell sorted and sequenced IgVRG from plasmablasts. Samples were collected prior to and one and two weeks following each immunization. Our initial analyses indicated that technical error, the variation introduced by biological sampling and standard sample preparation, resulted in skewed output, and we developed a model to better estimate quantitative values from Ig-seq. We also utilized unique molecular identifiers to correct for nucleotide errors and PCR over-amplification. Our analysis of IgVRG following AVA administration reveals that the population of peripheral PBs following primary immunization is not distinguishable from the pre-immune peripheral PB repertoire. These PBs have more somatic mutations than expected for newly activated and differentiated naïve B cells, and are unlikely to be vaccine-elicited. In contrast, PBs observed following the 2nd dose have low mutation frequencies that increase upon subsequent vaccination. These clones are more persistent than clones first observed following any other immunization, but still make up a very small proportion of the overall repertoire. At no time is the clonal repertoire consistently dominated by a few clones, and the total and plasmablast repertoires are highly transient, even after the elicitation of vaccine-specific antibodies. AVA immunization thus results in a polyclonal B cell response which is not dominated by one or a few highly specific, strongly-elicited clones. We conclude that primary immunization by AVA is not sufficiently immunogenic to elicit vaccine-responsive, class-switched PBs to the periphery, nor is complete AVA immunization able to sustain proliferation of individual clones, providing insight into why AVA may require regular boosts.

With the advent of non-culture based methods, researchers are now aware of the presence of a microbiome within the human lung. The constant presence of microorganisms requires a regulated immune system that can differentiate between infectious organisms and non-infectious commensals. B cells in the lungs are a key member of adaptive immunity by producing antigen specific antibodies as well as immune regulatory roles by secreting various cytokines. In this work, we utilized lung and blood samples paired by donor to test the theory that human lungs hold a unique B cell repertoire. By phenotypic analysis, we found a smaller proportion of CD19+, CD20+ B cells in the lung in respect to the blood. Of this initial pool of cells, a significant proportion were class-switched memory B cells (CS Bmem) and a much lower proportion were naïve B cells in the lung. We have also observed a greater proportion of the residency marker CD69 in CD27- B cells and CD27+ Bmems in the lungs. CS Bmem cells from lung or blood were sorted into a B cell culture system to generate monoclonal antibodies to attempt to analyze antigen-antibody binding through a protein array. Finally, we have analyzed the immunoglobulin variable region genes (IgVRGs) of CS Bmem that are CD69+ or CD69-. We have found that diversity from the unmutated common ancestor (UCA) in pulmonary CS Bmem was as similar as those in the blood. However, comparisons by branch length and mean branch to trunk ratios, we observed greater diversity in the lung compared to blood. We observed both gain and loss of CD69 expression within clones shared between the lungs and blood. The work presented here suggests a model where the lung retains specialized, mature Bmems within the tissue by the expression of CD69. Once stimulated by their antigen, the progeny born from the subsequent expansion would not only spread within the tissue, but also be capable of returning to circulation. This would form an expanded B cell presence at the local tissue level as well as in circulation and efficiently prevent the progress of a potential infection.

Montréal Trigonix inc. 2018