Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes

Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease affecting the related subcellular organelles platelet dense granules, lysosomes, and melanosomes. The mouse genes for HPS, pale ear and pearl, orthologous to the human HPS1 and HPS2 (ADTB3A) genes, encode a novel protein of unknown function and the beta(3)A subunit of the AP-3 adaptor complex, respectively. To test for in vivo interactions between these genes in the production and function of intracellular organelles, mice doubly homozygous for the 2 mutant genes were produced by appropriate breeding. Cooperation between the 2 genes in melanosome production was evident in increased hypopigmentation of the coat together with dramatic quantitative and qualitative alterations of melanosomes of the retinal pigment epithelium and choroid of double mutant mice. Lysosomal and platelet dense granule abnormalities, including hyposecretion of lysosomal enzymes from kidneys and depression of serotonin concentrations of platelet dense granules were likewise more severe in double than single mutants. Also, lysosomal enzyme concentrations were significantly increased in lungs of double mutant mice. Interaction between the 2 genes was specific in that effects on organelles were confined to melanosomes, lysosomes, and platelet dense granules. Together, the evidence indicates these 2 HPS genes function largely independently at the whole organism level to affect the production and function of all 3 organelles. Further, the increased lysosomal enzyme levels in lung of double mutant mice suggest a cause of a major clinical problem of HPS, lung fibrosis. Finally, doubly mutant HPS mice are a useful laboratory model for analysis of severe HPS phenotypes.     

In vivo expression of the B7 costimulatory molecule by subsets of antigen-presenting cells and the malignant cells of Hodgkin's disease

The B-lymphocyte/accessory-cell activation antigen B7 (BB1) has been shown in vitro to stimulate T-lymphocyte proliferation and cytokine production via CD28 present on the latter cells. In this study, benign lymphoid tissues, lymphomas, and extralymphoid inflammatory sites were examined immunohistochemically using anti-B7 and other relevant monoclonal antibodies. B7 was expressed by benign transformed germinal center B cells, as it was by B cells of follicular lymphomas. B7 was also expressed by a subpopulation (a mean of 31% to 65%) of macrophages and dendritic cells in a variety of lymphoid tissues. It was present in abundance on all macrophages constituting sarcoid granulomas in lymph nodes. In extralymphoid inflammation, 17% to 35% of macrophages expressed B7 only weakly. Cases of Hodgkin's disease showed expression of B7 by the majority of Reed-Sternberg cells or malignant mononuclear variants, a phenomenon that potentially contributes to the lymphocytic accumulation that is a feature of this condition. CD28+ T cells were seen in all areas where T cells were present. B7+ and CD28+ cells colocalized in, for example, lymphoid follicles, lymph node paracortex, sarcoid granulomas, and Hodgkin's disease tissue, indicating a potential for cellular interaction via these molecules at these sites.     

Colonic motility diagnosis based on colonic pressure

Manometry of the alimentary tract is a valuable and widely used means to evaluate and diagnose the function of the alimentary tract. However, the measurement can be inconvenient due to the invasive method used, and the many factors affecting results. Research on colonic pressure data is even more insufficient. This paper deals with colonic pressure data via an improved method ensuring that pressure data of the whole colon is available. The data is analysed based on the learning vector quantization (LVQ) method. Testing results show that this method distinguishes the normal data and the abnormal data, consistently with the original diagnoses. This method can serve as an assistant diagnosis of colonic motility and contributes to further research on colonic motility based on pressure data.     

B-cell lymphoma after angioimmunoblastic lymphadenopathy: a case with oligoclonal gene rearrangements associated with Epstein-Barr virus

We describe a patient with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), who subsequently developed large-cell immunoblastic lymphoma of B-cell immunophenotype. At the time of the initial diagnosis, histologic examination of an inguinal lymph node showed typical features of AILD, and there was no evidence of a monoclonal B-cell population by immunohistochemical analysis. In situ hybridization and Southern blot analysis for Epstein-Barr virus (EBV) were negative. At autopsy 2 years later, the patient had widespread lymph node and organ involvement by large-cell immunoblastic lymphoma of B-cell immunophenotype. Southern blot analysis performed on DNA extracted from lymph nodes, liver, and spleen showed two patterns of Ig heavy chain and kappa light chain gene rearrangements. The T-cell receptor beta chain gene was in the germline configuration. Analysis with an EBV terminal repeat region probe showed two clonal populations that paralleled the Ig gene rearrangement studies. Double-labeling immunohistochemistry and in situ hybridization confirmed the presence of EBV within the neoplastic B cells. The data support the hypothesis that EBV was not etiologically related to AILD in this case, and that EBV proliferation may occur after the onset of the disease. Further, the data suggest that some B-cell lymphomas that arise in the setting of AILD resemble EBV-associated B-cell lymphomas that arise in other immunodeficiency states.     

Fucoidin, but not yeast polyphosphomannan PPME, inhibits leukocyte rolling in venules of the rat mesentery

Leukocyte rolling in venules is inhibited by several sulfated polysaccharides, by antibodies to the leukocyte adhesion receptor L- selectin (LECAM-1), and by recombinant soluble L-selectin. The sulfated fucose polymer fucoidin and the polyphosphomannan PPME bind to L- selectin and inhibit L-selectin-mediated lymphocyte adhesion to lymph node high endothelial venules (LN-HEV). We investigated whether fucoidin and PPME also inhibit leukocyte rolling. Rolling leukocyte flux was determined by intravital microscopy in 47 venules (diameter 21 to 50 microns) of the rat mesentery with and without micro-infusion of each reagent through 8-microns glass micropipettes. Micro-infusion (1 mg/mL) or intravenous (IV) injection (25 mg/kg) of fucoidin, but not vehicle, reduced leukocyte rolling by greater than 90%. The half- effective concentration was approximately 2.5 micrograms/mL. Stroboscopic fluorescence video microscopy showed that fucoidin decreased the fraction of rolling leukocytes from 44% of all leukocytes passing the venules in control to less than 1%. PPME micro-infusion (1 mg/mL) or IV injection (14 mg/kg) did not reduce leukocyte rolling. Hence, leukocyte rolling differs from lymphocyte homing with respect to the effect of PPME. This may be related to fucoidin binding to L- selectin with greater affinity than PPME. Alternatively, inflamed venular endothelium may express a ligand for L-selectin different from that constitutively expressed on LN-HEV.     

Ischemia Modified Albumin for the assessment of patients presenting to the emergency department with acute chest pain but normal or non-diagnostic 12-lead electrocardiograms and negative cardiac troponin T

BACKGROUND: The diagnosis of myocardial ischemia in patients with acute chest pain at rest but non-diagnostic electrocardiograms (ECG) is problematic. Ischemia Modified Albumin (IMA) is a new biochemical marker of ischemia, which may be useful to characterise acute coronary syndrome (ACS) patients. METHODS: We studied 131 patients (mean age 58.5 years; 95 male) presenting to the emergency department with symptoms suggestive of ACS but with normal or non-diagnostic ECGs. Cardiac troponin T (cTnT) and IMA were measured within 3 h of last chest pain episode. Based on hospital diagnostic test results, patients were classified as having ACS or non-ischemic chest pain (NICP), by two independent cardiologists unaware of IMA results. RESULTS: Mean IMA levels (U/ml) were higher in patients with ACS (98.3+/-11) compared to patients with NICP (85.5+/-15); p<0.0001. IMA levels >93.5 U/ml demonstrated a sensitivity and specificity of 75% for the diagnosis of ACS; area under the receiver operator characteristic curve 0.78 (95% CI: 0.70-0.85). If we applied the manufacturer cutoff point of 85 U/ml, the sensitivity of IMA increased to 90.6% with a specificity of 49.3% (negative predictive value=84.6%). In combination with cTnT (6-12 h) (>0.05 ng/ml), the sensitivity increased to 92.2%. After multivariate analysis, IMA levels >85 U/ml (odds ratio=14.6 [95% CI 4.4-48.4]; p<0.0001), age and prior myocardial infarction were independent predictors of ACS. CONCLUSION: IMA may be a useful biomarker for the identification of ACS in patients presenting with typical acute chest pain but normal or non-diagnostic ECGs.     

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.B cells provide a critical line of defense from pathogenic infections through the production of highly specific antibodies. The initial stages of B cell development occur in the bone marrow, where hematopoietic stem cells undergo stepwise rearrangements of the genes encoding the B cell receptor (BCR) and changes in the expression of cell surface receptors (Hardy et al., 1991). Immature B cells egress the bone marrow and migrate to the spleen to complete their development, going through transitional stages. Mature follicular B cells then recirculate throughout the body in search for cognate antigen, continually entering secondary lymphoid organs, including the LNs and spleen. Specific recognition of antigen by the BCR provides the first signal required for B cell activation. Typically, a second signal is required for maximal activation and is provided by CD4⁺ helper T cells after the presentation of processed antigen on the B cell surface. These two signals in combination trigger the proliferation and differentiation of B cells, which go on to form antibody-secreting plasma cells and to establish germinal center responses for affinity maturation (Rajewsky, 1996).B cell activation in vivo is predominantly triggered by antigen on the surface of a presenting cell (Batista and Harwood, 2009). The prevalence of this mode of activation has brought about a reevaluation of the importance of the cytoskeleton, given that the recognition of tethered antigen requires considerable alteration in B cell morphology (Fleire et al., 2006). Antigen-induced BCR signaling leads to radical reorganization of the actin cytoskeleton resulting in the modification of the BCR dynamics at the cell surface (Hao and August, 2005; Treanor et al., 2010; Treanor et al., 2011). Moreover the binding of membrane-bound antigen to cognate BCR triggers a cascade of intracellular signaling events that induces actin-dependent spreading of the B cell across the antigen-containing surface (Weber et al., 2008; Sohn et al., 2008; Depoil et al., 2008). However the mediators that link BCR signaling with reorganization of the actin cytoskeleton are currently not well defined.Among actin regulators, the RhoGTPases are a highly conserved family that function as molecular switches by cycling between inactive GDP (guanosine diphosphate) and active GTP (guanosine triphosphate) bound states (Tybulewicz and Henderson, 2009). RhoGTPase activity is modulated by G-nucleotide exchange factors (GEF) that promote the formation of the GTP-bound state and binding to various effectors involved in actin reorganization. Conversely, GTPase-activating proteins (GAP) catalyze the hydrolysis of GTP and thereby switch off RhoGTPase activity. The importance of the RhoGTPases as a whole in the regulation of B cell responses is highlighted by the far-reaching consequences that impaired activity of several GEFs, such as Vav and DOCK8, has on humoral immune responses (Doody et al., 2001; Fujikawa et al., 2003; Randall et al., 2009; Zhang et al., 2009).The importance of Rho GTPases in B cell physiology has been well established. For example, RhoA has been shown to regulate BCR signaling by influencing inositol-3 phosphate synthesis and calcium signaling (Saci and Carpenter, 2005). Moreover, B cell–specific inactivation of both Rac1 and Rac2 leads to virtually complete absence of B cells (Walmsley et al., 2003), and inactivation of Rac1 results in defects in spreading in transitional cells (Brezski and Monroe, 2007). However, although the inactivation of Rac2 leads to defects in B cell adhesion and synapse formation, it is unclear whether these proteins are involved in actin-dependent spreading in mature B cells (Arana et al., 2008).Cdc42 has been little characterized in B cells, in spite of its proven chief role as an essential regulator of cell cycle (Johnson and Pringle, 1990), cell polarity (Etienne-Manneville, 2004), and actin cytoskeleton in other cellular systems. This is likely due, at least in part, to the reported mild phenotype of mice lacking Cdc42 in B cells (Guo et al., 2009) compared with the severe deficiencies observed in animals lacking Rac family members (Walmsley et al., 2003). However, the mild phenotype is somehow surprising given that Cdc42 directly or indirectly associates with Wiskott–Aldrich Syndrome Protein (WASp) and in complex with Arp2/3 regulates cytoskeleton remodeling (Symons et al., 1996; Aspenström et al., 1996; Kolluri et al., 1996). Importantly, mutations in WAS gene lead to a X-linked, recessive disease characterized by recurrent infections, abnormal lymphocyte function, as well as an increased risk for systemic autoimmunity (Derry et al., 1994; Sullivan et al., 1994). WASp deficient B cells play a primary role in driving autoimmunity (Becker-Herman et al., 2011). The Cdc42 effectors WASp and N-WASp have both been implicated the regulation of actin reorganization in response to BCR antigen engagement (Westerberg et al., 2012; Liu et al., 2013). Besides, expression of a dominant negative form of Cdc42 in B cells leads to alterations of the actin cytoskeleton (Westerberg et al., 2001). In addition, Cdc42 has been shown to play a role in the polarization and secretion of lysosomal protein involved in antigen extraction (Yuseff et al., 2011).Here, we used a strategy harnessing the mb1 promoter to generate mice with a selective and very effective deletion of Cdc42 in early B cell progenitors (Hobeika et al., 2006). Using this model, we demonstrated that Cdc42 plays an essential role in many aspects of B cell biology, including the formation of mature B cells and the establishment of antibody responses. We went on to dissect the underlying cause of the severe immunodeficiency of these mice and found that Cdc42-deficient B cells exhibit defects in BCR signaling and presentation of internalized antigen, leading to reduced B–T cell interactions and the absence of germinal center responses in vivo. Moreover, Cdc42-deficient B cells can normally proliferate and class switch when stimulated with CD40 or LPS, but they are completely impaired in their ability to differentiate into plasma cells. Together, these attributes render Cdc42-deficient mice unable to mount antibody responses after immunization with model antigen or viral infection, and highlight a fundamental role for this RhoGTPase in the regulation of B cell responses.