Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Patterns of antiretroviral use in the United States of America: analysis of three observational databases.

OBJECTIVE: To characterize patterns of antiretroviral use in HIV-infected patients and explore variation by patient characteristics and disease stage. METHODS: Three large patient databases recording information derived from routine clinical attendance were analyzed: HIV Insight (n = 10 873), Target Management Services (n = 2226) and Clinical Partners (n = 1505). Each database records the dates of starting and stopping individual antiretroviral agents over time, measurements of CD4 T-cell counts and HIV-RNA levels at approximately 6-monthly intervals, and the demographic characteristics of patients. The number, frequency and duration of different antiretroviral combinations over time and their relationship to stage of HIV-disease and demographic characteristics were explored. RESULTS: Over 2000 different combinations of antiretroviral agents are recorded. From 1987 onwards, the use of zidovudine increased, with 23% of patients receiving monotherapy by 1990. The majority of treated patients remained on monotherapy until the introduction of highly active antiretroviral therapy (HAART) in 1996. By 1999, the standard of care was HAART, with 84% of patients beginning antiretroviral therapy with HAART. Those of African American race (odds ratio 0.59) and funded by Medicaid (odds ratio 0.72) were significantly less likely to begin antiretroviral therapy on HAART. Until 1995, there was a significant decrease in CD4 T-cell count when starting antiretroviral therapy. No significant trend was observed in either CD4 T-cell count or viral load after this time. Those starting on HAART therapies were significantly less likely to stop or switch regimens than those on nucleoside reverse transcriptase inhibitor (NRTI)-only therapies (P < 0.001). CONCLUSIONS: Complex patterns of antiretroviral treatment are observed in this large population. Changes over time mirror the introduction of the new antiretroviral agents

2,5-Bis(1,1,3,3-tetramethylbutyl)thiophene

There are two independent molecules in the asymmetric unit of the title compound, C20H36S. Crystals are non-merohedrally twinned by twofold rotation about [001]. The bulky octyl groups of each molecule are on the same side of the thiophene plane and are approximately parallel. S&#8212;C distances are in the range 1.729&#8197;(4)&#8211;1.745&#8197;(3)&#8197;&#197;, and the C&#8212;S&#8212;C angles are 92.98&#8197;(18) and 93.08&#8197;(17)&#176;. The CH2 groups of the octyl groups are involved in weak C&#8212;H...S intramolecular interactions

Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P &lt; 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P &lt; 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade

Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade

Cytokine receptor IL27RA is an NF-kB-responsive gene involved in CD38 upregulation in multiple myeloma

Multiple myeloma (MM) shows constitutive activation of canonical and non-canonical nuclear factor-ĸB (NF-ĸB) signaling through genetic mutations or stimuli from the tumour microenvironment (TME). A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-ĸB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules IL-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the mRNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on normal long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B-cells in an IL-21-dependent in vitro PC-differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody (mAb) therapies by increasing CD38-expression on tumour cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared to normal PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME