Blood Coagulation Factor Fibrinogen in Tumor Pathogenesis of Central Nervous System B-Cell Lymphoma.

Central nervous system (CNS) lymphoma is an extranodal non-Hodgkin B-cell lymphoma characterized by malignant lymph tissue arising in the brain or spinal cord, associated with inflammation and blood-brain barrier (BBB) disruption. Although BBB disruption is known to occur in patients with CNS lymphoma, a direct link between these two has not been shown. Herein, abundant deposition of the blood coagulation protein fibrinogen around B-cell lymphoma was detected in CNS lymphoma patients and in the CNS parenchyma in an orthotopic mouse model. Functional enrichment analysis of unbiased cerebrospinal fluid proteomics of CNS B-cell lymphoma patients showed that coagulation protein networks were highly connected with tumor-associated biological signaling pathways. In vivo two-photon imaging demonstrated that lymphoma growth was associated with BBB disruption, and in vitro experiments identified a role for fibrinogen in promoting lymphoma cell adhesion. Overall, these results identify perivascular lymphoma clustering at sites of fibrinogen deposition, and suggest that fibrinogen may be a target for pharmacologic intervention in metastatic B-cell lymphoma associated with BBB disruption.

CXCL13 plus interleukin 10 is highly specific for the diagnosis of CNS lymphoma.

Establishing the diagnosis of focal brain lesions in patients with unexplained neurologic symptoms represents a challenge. The goal of this study is to provide evidence supporting functional roles for CXC chemokine ligand (CXCL)13 and interleukin (IL)-10 in central nervous system (CNS) lymphomas and to evaluate the utility of each as prognostic and diagnostic biomarkers. We demonstrate for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXCL13 and CXCL12 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions. Expression of the activated form of Janus kinase 1 supported a role for IL-10 in prosurvival signaling. We determined the concentration of CXCL13 and IL-10 in CSF of CNS lymphoma patients and control cohorts including inflammatory and degenerative neurologic disease in a multicenter study involving 220 patients. Bivariate elevated CXCL13 plus IL-10 was 99.3% specific for primary and secondary CNS lymphoma, with sensitivity significantly greater than reference standard CSF tests. These results identify CXCL13 and IL-10 as potentially important biomarkers of CNS lymphoma that merit further evaluation and support incorporation of CXCL13 and IL-10 into diagnostic algorithms for the workup of focal brain lesions in which lymphoma is a consideration.

Accelerated pathological and clinical nephritis in systemic lupus erythematosus-prone New Zealand Mixed 2328 mice doubly deficient in TNF receptor 1 and TNF receptor 2 via a Th17-associated pathway.

TNF-alpha has both proinflammatory and immunoregulatory functions. Whereas a protective role for TNF administration in systemic lupus erythematosus (SLE)-prone (New Zealand Black x New Zealand White)F(1) mice has been established, it remains uncertain whether this effect segregates at the individual TNFR. We generated SLE-prone New Zealand Mixed 2328 mice genetically deficient in TNFR1, in TNFR2, or in both receptors. Doubly-deficient mice developed accelerated pathological and clinical nephritis with elevated levels of circulating IgG anti-dsDNA autoantibodies and increased numbers of CD4(+) T lymphocytes, especially activated memory (CD44(high)CD62L(low)) CD4(+) T cells. We show that these cells expressed a Th17 gene profile, were positive for IL-17 intracellular staining by FACS, and produced exogenous IL-17 in culture. In contrast, immunological, pathological, and clinical profiles of mice deficient in either TNFR alone did not differ from those in each other or from those in wild-type controls. Thus, total ablation of TNF-alpha-mediated signaling was highly deleterious to the host in the New Zealand Mixed 2328 SLE model. These observations may have profound ramifications for the use of TNF and TNFR antagonists in human SLE and related autoimmune disorders, as well as demonstrate, for the first time, the association of the Th17 pathway with an animal model of SLE.

Cutaneous T-Cell Lymphoma PDX Drug Screening Platform Identifies Cooperation between Inhibitions of PI3Kα/δ and HDAC.

Cutaneous T-cell lymphoma is a form of non-Hodgkin lymphoma that manifests initially in the skin and disseminates systemically as the disease progresses. Mycosis fungoides and Sézary syndrome are the most common subtypes of cutaneous T-cell lymphoma. Advanced mycosis fungoides and Sézary syndrome are life threatening with few treatment options. We searched for new agents by high-throughput screening of selected targeted compounds and identified high-value targets, including phosphatidylinositol 3-kinase (PI3K) and cyclin-dependent kinases. To validate these hits from the screen, we developed patient-derived xenograft mouse models that recapitulated the cardinal features of mycosis fungoides and Sézary syndrome and maintained histologic and molecular characteristics of their clinical counterparts. Importantly, we established a blood-based biomarker assay using tumor cell-free DNA to measure systemic tumor burden longitudinally in living mice during drug therapy. A PI3K inhibitor, BKM120, was tested in our patient-derived xenograft model leading to disease attenuation and prolonged survival. Isoform-specific small interfering RNA knockdowns and isoform-selective PI3K inhibitors identified PI3K-δ as required for tumor proliferation. Additional studies showed a synergistic combination of PI3K-α/δ inhibitors with histone deacetylase inhibitors. The strong preclinical efficacy of this potent combination against multiple patient-derived xenograft models makes it an excellent candidate for further clinical development.

Subtoxic levels hydrogen peroxide-induced production of interleukin-6 by retinal pigment epithelial cells.

PURPOSE: To study the effect of subtoxic levels of hydrogen peroxide (H(2)O(2)) on the expression and release of interleukin-6 (IL-6) by cultured retinal pigment epithelial (RPE) cells and to explore the relevant signal pathways. METHODS: Cultured human RPE cells were stimulated with various subtoxic concentrations of H(2)O(2) for different periods. Conditioned medium and cells were collected. IL-6 in the medium and IL-6 mRNA in the collected cells were measured using an IL-6 enzyme-linked immunosorbent assay kit and reverse transcriptase polymerase chain reaction, respectively. Nuclear factor-kappaB (NF-κB) in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK) in cells cultured with and without H(2)O(2) were measured by NF-κB and MAPK enzyme-linked immunosorbent assay kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-κB (BAY11-7082) were added to the cultures before the addition of H(2)O(2) to test their effects(.) RESULTS: Subtoxic levels of H(2)O(2) (100 µM and less) increased the IL-6 mRNA level and the release of IL-6 protein by the cultured human RPE cells in a dose- and time-dependent manner. This was accompanied by an increase of NF-κB in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cell lysates, particularly in the p38 and NF-κB. The NF-κB inhibitor decreased the H(2)O(2)-induced expression of IL-6. The p38 inhibitor, but not the ERK or JNK inhibitor, completely abolished H(2)O(2)-induced expression of IL-6 by RPE cells. The p38 inhibitor also abolished the increase of NF-κB in nuclear extracts in cells treated with H(2)O(2). CONCLUSIONS: H(2)O(2) stimulated the production of IL-6, a key factor in the modulation of immune responses, inflammatory processes, and the occurrence of autoimmune diseases, which recently has been documented to be increased in age-related macular degeneration (AMD). This may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in AMD and may provide a novel target for the treatment of AMD.

Depression is an early disease manifestation in lupus-prone MRL/lpr mice.

Many lupus patients develop neuropsychiatric manifestations, including cognitive dysfunction, depression, and anxiety. However, it is not clear if neuropsychiatric lupus is a primary disease manifestation, or is secondary to non-CNS disease. We found that MRL/lpr lupus-prone mice exhibited significant depression-like behavior already at 8 weeks of age, despite normal visual working memory, locomotor coordination and social preference. Moreover, depression was significantly correlated with titers of autoantibodies against DNA, NMDA receptors and cardiolipin. Our results indicate that lupus mice develop depression and CNS dysfunction very early in the course of disease, in the absence of substantial pathology involving other target organs.

TNF-like weak inducer of apoptosis (TWEAK) induces inflammatory and proliferative effects in human kidney cells.

Members of the TNF-ligand and receptor superfamilies are important in the pathogenesis of lupus nephritis, a major cause of mortality and morbidity in SLE. TWEAK, a member of the TNF-ligand superfamily, is markedly increased in urine from patients with active lupus nephritis, and urinary TWEAK levels significantly correlate with renal disease activity. To support a possible role of TWEAK in the pathogenesis of lupus nephritis and other inflammatory nephritides, we examined the effects of TWEAK in human kidney mesangial cells, podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. We found that TWEAK induces human kidney cells to express multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1alpha, ICAM-1, and VCAM-1. Cytokine production is mediated through NF-kappaB activation, and is inhibited by anti-TWEAK monoclonal antibodies. TWEAK stimulated chemokines induced migration of human PBMC, particularly monocytes/macrophages. Furthermore, we found that TWEAK promotes kidney infiltration of inflammatory cells, and stimulates proliferation of kidney cells in vitro and in vivo. Thus, TWEAK may play an important pathogenic role in the development of glomerulonephritis by promoting a local inflammatory environment and inducing kidney cell proliferation. Blocking TWEAK/Fn14 interactions may be a promising therapeutic target in immune-mediated renal diseases.

Autoantibodies in lupus: culprits or passive bystanders?

Several autoantibodies are culprits in the pathogenesis of organ damage in systemic lupus erythematosus, by means of established or postulated mechanisms, whereby inducing a perturbation of cell structure and function, with consequent tissue-organ impairment. Common autoantibody-mediated mechanisms of damage include cell surface binding with or without cytolysis, immune complex-mediated damage, penetration into living cells, binding to cross-reactive extracellular molecules. Experimental data from both murine models and humans have recently clarified the key role of autoantibodies in severe organ involvements, including nephritis, neuropsychiatric (NP) dysfunction, and cerebrovascular disease (CVD). In lupus nephritis early and late phases are distinguishable and mediated by different processes in which anti-chromatin antibodies are both inducing and perpetuating agents, by immune-complex formation and massive deposition in mesangial matrix at first, and in glomerular basement membrane at end-stage. Also NP abnormalities occur very early, much earlier than other systemic manifestations, and exacerbate with the increase in autoantibody titers. Among the autoantibodies mainly implicated in neurolupus, anti-ß2 glycoprotein I (ß2GPI) antibodies are preferentially involved in focal NP events which are a consequence of non-inflammatory microangiopathy; otherwise, anti-ribosomal P protein antibodies and N-methyl-d-aspartate receptor (NMDAR) antibodies cause diffuse NP events through a direct cytotoxic effect on neuronal cells at specific brain zones.

Sex and autoantibody titers determine the development of neuropsychiatric manifestations in lupus-prone mice.

Emotional disturbances are among the most common neuropsychiatric manifestations of SLE, a systemic autoimmune disease with a strong female predominance. In this study, we evaluated young MRL/lpr mice, directly comparing males and females. MRL/lpr females exhibited significant depression as early as 5 weeks (at which time elevated levels of autoantibodies were already present), as compared to MRL/lpr males, where depression was noted only at 18 weeks. Depression was significantly correlated with autoantibodies against nuclear antigens, NMDA receptor, and ribosomal P. Our results are consistent with a primary role of autoantibodies in the pathogenesis of early neuropsychiatric deficits in this lupus model, which translate into gender-based differences in clinical phenotype.

Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-gamma-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-alpha + IL-1beta. CXCL11/interferon-inducible T cell alpha chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Ikappa-B, while pretreatment with an Ikappa-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-kappaB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.