Rituximab and obinutuzumab differentially hijack the B-cell receptor and NOTCH1 signaling pathways

The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B-cell death. To characterize the signaling processes responsible for improved B-cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B-cell receptor. While both antibodies induce strong ERK and MYC activation sufficient to promote cell cycle arrest and B-cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS

Visualization of genomic aberrations using Affymetrix SNP arrays

doi:10.1093/bioinformatics/btl60

The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis

Hemorrhagic Shock Induces a Rapid Transcriptomic Shift of the Immune Balance in Leukocytes after Experimental Multiple Injury

The immune response following trauma represents a major driving force of organ dysfunction and poor outcome. Therefore, we investigated the influence of an additional hemorrhagic shock (HS) on the early posttraumatic immune dysbalance in the whole population of blood leukocytes. A well-established murine polytrauma (PT) model with or without an additional pressure-controlled HS (mean arterial pressure of 30 mmHg (±5 mmHg) for 60 mins, afterwards fluid resuscitation with balanced electrolyte solution four times the volume of blood drawn) was used. C57BL/6 mice were randomized into a control, PT, and PT + HS group with three animals in each group. Four hours after trauma, corresponding to three hours after induction of hemorrhage, RNA was isolated from all peripheral blood leukocytes, and a microarray analysis was performed. Enrichment analysis was conducted on selected genes strongly modulated by the HS. After additional HS in PT mice, the gene expression of pathways related to the innate immunity, such as IL-6 production, neutrophil chemotaxis, cell adhesion, and toll-like receptor signaling was upregulated, whereas pathways of the adaptive immune system, such as B- and T-cell activation as well as the MHC class II protein complex, were downregulated. These results demonstrate that an additional HS plays an important role in the immune dysregulation early after PT by shifting the balance to increased innate and reduced adaptive immune responses

Increased migratory potential of CD69-deficient CD4 T cells to the mucosal intestinal tissues <i>in vivo</i>.

<p>CD4 T cells were enriched from the spleen of red fluorescent DsRed or CD69^−/− animals, both on the CD45.2⁺ B6 background. CD69-deficient CD4 T cells were labelled with green fluorescent CFSE. These cells were mixed in the approximate ratio 1∶1 and injected i.v. into CD45.1⁺ B6 mice. 18 or 72 h later cells were isolated from blood, mesenteric lymph nodes (MLN), small intestinal lamina propria (siLP) and colonic lamina propria (cLP) of the hosts and analyzed for the surface expression of CD4, CD45.2, DsRed and CFSE by multicolour flow cytometry (FCM). Numbers of DsRed and CFSE expressing CD45.2⁺ CD4⁺ cells recovered 18 h (<b>A</b>) or 72 h (<b>D</b>) after the transfer from the host tissues were determined with FCM and presented as mean (± SEM) total number of recovered cells per tissue for four mice analyzed. N.S. – not statistically significant; *p≤0.05. Homing index (HI) for every tissue 18 h (<b>B</b>) or 72 h (<b>E</b>) post-injection is calculated as: HI = number of CD4⁺CD45.2⁺CFSE⁺ cells/number of CD4⁺CD45.2⁺DsRed⁺ cells : IR (where IR is input ratio calculated before the injection as: IR = number of CD4⁺CD45.2⁺CFSE⁺ cells/number of CD4⁺CD45.2⁺DsRed⁺ cells). This value was normalized to the HI in the blood, so the potential retention of the injected cells in some of the periphery organs is eliminated. Mean (± SEM) of blood-normalized HI per tissue for four mice is presented. The deviation from the theoretical mean (TM = 1) was assessed (*p≤0.05). <b>C.</b> 18 h after the cell transfer sections of small intestine and colon of the host were stained with Alexa Fluor 350-conjugated wheat germ agglutinin (blue) analyzed for the number of green (CFSE⁺ CD69^−/−) and red (DsRed⁺ B6) CD4 T cells by confocal microscope (original magnification×40).</p