Automatic tracking of rolling leukocytes in vivo.

The analysis of instantaneous and average rolling leukocyte velocity is crucial to the study of inflammatory disease. In order to record features associated with leukocyte rolling, the leukocyte position must be tracked, typically by manual observation. Automated tracking of leukocytes is possible for in vitro studies, but not for recordings resulting from intravital experiments. Therefore, we have designed and implemented an image processing system for automated tracking of rolling leukocytes in vivo. The novel image processing techniques used in the tracking system successfully address the four major problems associated with tracking cells in vivo: background movement, severe image noise and clutter, cell deformation and contrast change, and occlusion of the target cell by other structures. We have tested the system in two experimental protocols in which leukocyte rolling is observed in venules of the mouse cremaster muscle with and without TNF-alpha treatment. The automated tracking system was validated by comparing automatically generated displacement and velocity data with data from the same recordings collected manually. The root mean squared error between the computed displacements and the manually measured displacements was less than 12% of the average displacement in TNF-alpha-treated venules. The average velocity error was also less than 12%. For untreated venules, the computed and measured displacements and velocities had an RMSE of less than 8%. The automated tracking system allows one, for the first time, to reliably track rolling leukocytes in vivo, thus eliminating possible investigator bias and increasing throughput.     

Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules

At the onset of the inflammatory process, leukocytes roll along venular but not arteriolar walls before they firmly attach and emigrate. To test whether differences in hydrodynamic flow conditions are responsible for the preferential occurrence of leukocyte rolling in venules, we varied wall shear rate, gamma w, between 30 and 2,000 sec-1 by selective micro-occlusion of side branches in venules and arterioles (diameter, 20-37 microns) of the exposed mesentery of anesthetized rats. In venules, 39% (range, 6-77%) of all passing leukocytes were found interacting with the endothelium (rolling), whereas this fraction was only 0.6% in arterioles. The fraction of rolling leukocytes in venules decreased from 49 +/- 13% at gamma w less than 100 sec-1 (N = 12) to 24 +/- 13% at gamma w greater than 400 sec-1 (N = 12). Mean leukocyte rolling velocity in venules increased with gamma w, but the most frequent rolling velocity class was 20-40 microns/sec at all shear rates. In arterioles, even prolonged (up to 90 minutes) conditions of reduced flow (gamma w less than 150 sec-1) did not induce leukocyte rolling. Radial distribution of freely flowing leukocytes not different in arterioles and venules. The data indicate that hemodynamic factors are not responsible for the difference of leukocyte adhesion between arterioles and venules. The venular endothelium appears to be specialized to support leukocyte adhesion during inflammation. This finding correlates with reports on preferential expression of various endothelial-leukocyte adhesion molecules on venular endothelial cells.     

Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells.

Adhesion of isolated human polymorphonuclear granulocytes (PMNs) to five different phenotypes of cultured microvascular endothelial cells derived from bovine corpora lutea was investigated by measuring the myeloperoxidase content of cell lysates. Untreated and interleukin 1 (IL-1) -pretreated confluent monolayers were overlaid with unstimulated and phorbol ester (PMA)-stimulated PMNs in the absence and presence of the monoclonal antibody IB4 recognizing and functionally blocking beta 2 (CD18) of the leukocyte integrins. Unstimulated PMN adhesion was highest on type 4, followed by type 3 and 5 endothelial cells. This adhesion was not inhibited by treatment with IB4. IL-1 pretreatment of endothelial cells resulted in a significant increase of PMN adhesion on types 1, 2, and 4, most of which was also beta 2 integrin-independent. PMA-stimulation of PMNs increased adhesion to maximal values on cell types 1 and 5, which was largely blocked by IB4. Type 2 endothelial cells supported significantly less PMA-stimulated PMN adhesion than all other types. In the presence of IB4, adhesion of PMNs to untreated and IL-1-pretreated type 3 and 4 endothelial cells was significantly reduced by PMA. This reduction of beta 2 integrin-independent adhesion by PMA stimulation is compatible with possible shedding of the lectin-like leukocyte adhesion molecule, L-selectin, from PMNs. Differential PMN adhesion may reflect distinctive expression of endothelial adhesion molecules in different phenotypes of microvascular endothelial cells. Endothelial specialization within the microcirculation may have important functional consequences for the inflammatory response in vivo.     

Identification of somatic JAK1 mutations in patients with acute myeloid leukemia

Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1^T478S, and JAK1^V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis.     

DNA Methylation Variants at HIF3A Locus,B-Vitamin Intake,and Long-term Weight Change: Gene-Diet Interactions in Two U.S. Cohorts

The first epigenome-wide association study of BMI identified DNA methylation at an HIF3A locus associated with BMI. We tested the hypothesis that DNA methylation variants are associated with BMI according to intake of B vitamins. In two large cohorts, we found significant interactions between the DNA methylation–associated HIF3A single nucleotide polymorphism (SNP) rs3826795 and intake of B vitamins on 10-year changes in BMI. The association between rs3826795 and BMI changes consistently increased across the tertiles of total vitamin B₂ and B₁₂ intake (all P for interaction <0.01). The differences in the BMI changes per increment of minor allele were −0.10 (SE 0.06), −0.01 (SE 0.06), and 0.12 (SE 0.07) within subgroups defined by increasing tertiles of total vitamin B₂ intake and −0.10 (SE 0.06), −0.01 (SE 0.06), and 0.10 (SE 0.07) within subgroups defined by increasing tertiles of total vitamin B₁₂ intake. In two independent cohorts, a DNA methylation variant in HIF3A was associated with BMI changes through interactions with total or supplemental vitamin B₂, vitamin B₁₂, and folate. These findings suggest a potential causal relation between DNA methylation and adiposity.     

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

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.     

Establishing and managing a periodontal biobank for research: the sharing of experience

Periodontal bio‐repositories, which allow banking of clinically validated human data and biological samples, provide an opportunity to derive biomarkers for periodontal diagnosis, prognosis and therapeutic activities which are expected to improve patient management. This article presents the establishing of the Malaysian Periodontal Database and Biobank System (MPDBS) which was initiated in 2011 with the aim to facilitate periodontal research. Partnerships were established with collaborating centres. Policies on specimen access, authorship and acknowledgement policies were agreed upon by all participating centres before the initiation of the periodontal biobank. Ethical approval for the collection of samples and data were obtained from institutional ethics review boards. A broad‐based approach for informed consent was used, which covered areas related to quality of life impacts, genetics and molecular aspects of periodontal disease. Sample collection and processing was performed using a standardized protocol. Biobanking resources such as equipment and freezers were shared with the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS). In the development of the MPDBS, challenges that were previously faced by the MOCDTBS were considered. Future challenges in terms of ethical and legal issues will be faced when international collaborations necessitate the transportation of specimens across borders.