ALBERT

Description: Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Description: We show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α5β1, α3β1, α6β1, and α1β1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Description: To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic growth factors, reproducible differentiation was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein–positive (GFP+) and GFP– hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately 1:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed differentiation of hESCs.

Description: Purpose Ovarian cancer is the most lethal gynecologic cancer. Chemotherapy, the standard of care, has hematologic toxicity, primarily neutropenia. G-CSF is currently used to support white blood cell (WBC) and absolute neutrophil counts (ANC). Prior clinical trials from China suggest that acupuncture could ameliorate chemotherapy-induced leukopenia; the proposed mechanism is an increase in G-CSF levels. In the current study, we investigated the effect of acupuncture, administered during myelosuppressive therapy, on WBC and ANC counts in ovarian cancer patients. Patients and methods Twenty-one newly diagnosed or recurrent ovarian cancer patients were randomized to receive active versus sham acupuncture while undergoing standard IV platinum and taxane-containing chemotherapy. A standardized protocol with 9 acupuncture points was employed with manual and electroacupuncture stimulation. The frequency of acupuncture treatment was 2–3 times per week for a total of 10 sessions, starting 1 week before the 2nd cycle of chemotherapy. WBC and ANC counts were checked weekly at five time points. Serum G-CSF was collected four times during the study. Results Of 587 patients screened, 21 patients were enrolled and received either acupuncture or sham treatment. Patients in both the active and control arms had similar patient characteristics and treatment. Both median WBC and ANC values at nadir in the acupuncture arm were higher than in the control arm, but the differences were not statistically significant, after adjusting for the baseline difference. However, the median WBC in the acupuncture arm at recovery was statistically significantly higher than the control arm, after adjustment (8,600 cell/μL, range: 4,800–12,000 vs. 4,400 cell/μL range: 2,300–10,000) (p=0.045). The recovering median ANC in the patients receiving acupuncture also was higher, but this difference was not statistically significant (p=0.094). The median serum G-CSF at baseline for patients in the active vs. control arm was similar (37.3 pg/mL, range 28.6–393.3 vs. 32.0, range 11.8–211.3, respectively) (p=0.291). At the second time point, the 1st day of the 2nd cycle, the acupuncture group had a higher G-CSF value than the control group (p=0.121). At nadir, the acupuncture group still had a slightly higher G-CSF value than in the control group (p=0.796). However, at the recovery day, the 1st day of 3rd cycle, the G-CSF value in the acupuncture group was lower than in the control arm (p=0.729). No statistical significance in G-CSF value was found at each time point between the two groups. Conclusion The acupuncture protocol used in this study was feasible and safe. We report trends of higher WBC and ANC values during one cycle of myelosuppressive chemotherapy in ovarian cancer patients, suggesting a potential myeloprotective effect of acupuncture. However, current data do not support an acupuncture effect on G-CSF production. These findings warrant a larger study to explore the observed clinical trends and other potential underlying mechanisms.

Description: Backround: Arsenic trioxide (ATO) has been shown to be synergistic with melphalan both in vitro and in vivo. We conducted a phase I/II trial to determine the safety and efficacy of a combination of arsenic trioxide, melphalan and ascorbic acid (AA) as preparative regimen in patients undergoing high-dose therapy (HDT) and autologous hematopoietic progenitor cell transplantation for multiple myeloma (MM). We also assessed the impact ATO levels on melphalan pharmacokinetics (PK), engraftment and toxicity. Methods: Forty-eight patients with secretory myeloma (23 females, 25 males; median age: 54, range: 3570) were treated between 4/04 and 8/05. All patient received melphalan 100 mg/m2 IV on days -4 and -3 and AA 1000 mg/day IV on days -9 to -3. Patients were randomized to 3 arms; no ATO (arm 1), ATO 0.15 mg/kg IV on days -9 to -3 (arm 2) and ATO 0.25 mg/kg IV on days -9 to -3 (arm 3). Twelve patients had disease progression or relapse after a prior autograft. Median CD34 cells dose infused was 4.5 x 106/kg (range 2.3–10.9). Results: Patients in all 3 arms were evenly matched. With a median F/U of 14.0 months (range 6–25) post autograft, no dose-limiting toxicity or non-relapse mortality was seen. Toxicity was limited to grade I or II nausea, vomiting and diarrhea. Median ATO levels on day 0 in arms 1, 2 and 3 were 0.2, 26.3 and 46.2 ng/ml, respectively. Melphalan PK was not altered by ATO pretreatment. Median time to neutrophil engraftment (ANC 〉500/ dl) was 9 days. There were no engraftment failures or delays in the ATO arms. CR rate for the entire group was 23%, and total response rate (CR + PR) was 75%. 1-year Progression-free survival (PFS) and overall survival (OS) were 75% and 95%, respectively. There was no significant difference in CR, RR, PFS or OS between the 3 arms (p = 0.9, 0.9, 0.4 and 0.6, respectively). A prior autologous transplant (p = 0.02) and abnormal cytogenetics at transplant (p = 0.04) were associated with a significantly shorter remission. Conclusions: ATO + melphalan + ascorbic acid is a safe, effective and well tolerated preparative regimen for patients with multiple myeloma undergoing an autotransplant. A longer follow up is needed to assess the impact of ATO on progression-free and overall survival.

Description: DLBCL is a curable subtype of non-Hodgkin lymphoma, although a significant number of patients do not achieve a remission or they relapse with conventional chemotherapy. While clinical variables (e.g., IPI), tumor (somatic) genetic alterations, and gene expression profiling have all been shown to predict outcome, there remains a need for additional prognostic biomarkers. One understudied class of biomarkers is host genetic background. We evaluated the hypothesis that germline variability in 73 SNPs from 44 candidate immune genes was associated with overall survival in DLBCL. We addressed this hypothesis in 365 DLBCL patients aged 20–70 years who participated in a population-based case-control study conducted from 1998–2000 (prior to the use of R-CHOP) through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles. Germline DNA was extracted from a venous blood sample or mouthwash buccal cell sample, which was collected a median of 4.8 months after diagnosis in this population-based study. All genotyping was conducted at the National Cancer Institute Core Genotyping Facility using the Taqman platform, and was successful in over 95% of the DNA samples for the SNPs evaluated. Histology, stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site in the spring of 2005. Cox proportional hazards analysis was used to evaluate the association between individual SNPs, adjusted for age, demographic and clinical factors. Parallel modeling strategies were used to identify the best summary multi-SNP risk score to predict survival. At a median follow-up of 56 months (range, 27-78 months) for surviving patients, there were 96 deaths in 365 patients (26%). In multivariate modeling, SNPs in IL1A (rs1800587; HRCT/TT=1.90, 1.26–2.87), IL6 (rs1800795; HRGG=1.48, 0.99–2.23), IL-10 (rs1800896; HRAG/GG=1.48, 0.91–2.38), and IFNGR2 (rs2070385; HRAG/GG=1.35, 0.86–2.11) were the strongest and most robust predictors of overall survival. A summary score of the number of deleterious genotypes from these four genes in combination with clinical and demographic variables was strongly associated with survival (p=9.3 x 10−12); Kaplan-Meier 5-year survival estimates for low, intermediate, and high risk patients were 89%, 68%, and 47% respectively. In conclusion, host genetic background as measured by germline polymorphisms in immune genes including IL1A, IL6, IL10, and IFNGR2 were associated with overall survival in DLBCL after accounting for clinical and demographic factors. These promising results require confirmation and need further evaluation in patients treated with R-CHOP in conjunction with tumor and other prognostic biomarkers.

Description: The most frequently recurring translocations in mucosa-associated lymphoid tissue (MALT) B-cell non-Hodgkin lymphoma, t(11;18)(q21;q21) and t(14;18)(q32; q21), lead to formation of an API2-MALT1 fusion or IgH-mediated MALT1 overexpression. Various approaches have implicated these proteins in nuclear factor κB (NF-κB) signaling, but this has not been shown experimentally in human B cells. Immunohistochemistry showed that MALT1 is predominantly expressed in normal and malignant germinal center B cells, corresponding to the differentiation stage of MALT lymphoma. We expressed MALT1 and apoptosis inhibitor-2 API2/MALT1 in human B-cell lymphoma BJAB cells and found both transgenes in membrane lipid rafts along with endogenous MALT1 and 2 binding partners involved in NF-κB signaling, B-cell lymphoma 10 (BCL10) and CARMA1 (caspase recruitment domain [CARD]-containing membrane-associated guanylate kinase [MAGUK] 1). API2-MALT1 and exogenous MALT1 increased constitutive NF-κB activity and enhanced IκB kinase (IKK) activation induced by CD40 stimulation. Both transgenes protected BJAB cells from FAS (CD95)-induced death, consistent with increases in NF-κB cytoprotective target gene expression, and increased their proliferation rate. Expression of a dominant-negative IκBα mutant showed that these survival and proliferative advantages are dependent on elevated constitutive NF-κB activity. Our findings support a model in which NF-κB signaling, once activated in a CD40-dependent immune response, is maintained and enhanced through deregulation of MALT1 or formation of an API2-MALT1 fusion.

Description: Gram-negative bacterial endotoxemia may lead to the pathological increase of vascular permeability with systemic vascular collapse, a vascular leak syndrome, multiple organ failure (MOF), and/or shock. Previous studies demonstrated that C1 inhibitor (C1INH) protects mice from lipopolysaccharide (LPS)–induced lethal septic shock via a direct interaction with LPS. Here, we report that C1INH blocked the LPS-induced increase in transendothelial flux through an endothelial monolayer. In addition, LPS-mediated detachment of cultured endothelial cells was prevented with C1INH. C1INH also inhibited LPS-induced endothelial cell apoptosis as demonstrated by suppression of DNA fragmentation and annexin V expression. As illustrated by laser scanning confocal microscopy, C1INH completely blocked the binding of fluorescein isothiocyanate (FITC)–LPS to human umbilical vein endothelial cells (HUVECs). C1INH protected from localized LPS-induced increased plasma leakage in C57BL/6J mice and in C1INH-deficient mice. Local vascular permeability in response to LPS was increased to a greater extent in C1INH-deficient mice compared with wild-type littermate controls and was reversed by treatment with C1INH. Systemic administration of LPS to mice resulted in increased vascular permeability, which was reduced by C1INH. Therefore, these studies demonstrate that C1INH, in addition to its role in suppression of LPS-mediated macrophage activation, may play an important role in the prevention of LPS-mediated increased vascular permeability, endothelial cell injury, and multiple organ failure.

Description: Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial “tip cell” phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFκB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFκB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Description: A role for genetic susceptibility in non-Hodgkin lymphoma (NHL) is supported by the accumulating evidence of common genetic variations altering NHL risk. However, the pattern of NHL heritability remains poorly understood. We conducted a pooled analysis of 10 211 NHL cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph) to evaluate NHL risk among those with hematopoietic malignancies in first-degree relatives. Odds ratios (ORs) and 95% confidence intervals (CIs) of NHL and its subtypes were estimated from unconditional logistic regression models with adjustment for confounders. NHL risk was elevated for individuals who reported first-degree relatives with NHL (OR = 1.5; 95% CI = 1.2-1.9), Hodgkin lymphoma (OR = 1.6; 95% CI = 1.1-2.3), and leukemia (OR = 1.4; 95% CI = 1.2-2.7). Risk was highest among individuals who reported a brother with NHL (OR = 2.8; 95% CI = 1.6-4.8) and was consistent for all NHL subtypes evaluated. If a first-degree relative had Hodgkin lymphoma, NHL risk was highest if the relative was a parent (OR = 1.7; 95% CI = 1.0-2.9). If a first-degree relative had leukemia, NHL risk was highest among women who reported a sister with leukemia (OR = 3.0; 95% CI = 1.6-5.6). The pattern of NHL heritability appeared to be uniform across NHL subtypes, but risk patterns differed by specific hematopoietic malignancies and the sex of the relative, revealing critical clues to disease etiology.