Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines

The current study shows that a clonal derivative of the Jurkat cell line up-regulates both the avidity and density of the α 6/β1 receptor in response to phorbol 12-myristate 13-acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up-regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexaminde, can be blocked by monoclonal antibodies (Mabs) to the β1 and α 5 subunits of the β1 family of integrins, and is not associated with increased expression of the α 5 or β1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48–72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the β1 and α 6 subunits, and is associated with increased expression of the α 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB-2, CEM, Molt-4, and HPB-ALL T-lymphoblastic cell lines also up-regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up-regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4–120 hours of treatment. The most mature lines (Jurkat and HPB-ALL) up-regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt-4, and HSB-2). In summary, clonal derivatives of the Jurkat cell line up-regulated attachment to laminin through protein kinase dependent increases in α /β1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T-lymphoblastic cell lines. © 1993 Wiley-Liss, Inc.     

Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.     

Associations of physical activity with gut microbiota in pre-adolescent children

[Purpose] To determine whether physical activity (PA), primarily the recommended 60 minutes of moderate-to-vigorous PA, is associated with gut bacterial microbiota in 10-year-old children.[Methods] The Block Physical Activity Screener, which provides minutes/day PA variables, was used to determine whether the child met the PA recommendations. 16S rRNA sequencing was performed on stool samples from the children to profile the composition of their gut bacterial microbiota. Differences in alpha diversity metrics (richness, Pielou’s evenness, and Faith’s phylogenetic diversity) by PA were determined using linear regression, whereas beta diversity (unweighted and weighted UniFrac) relationships were assessed using PERMANOVA. Taxon relative abundance differentials were determined using DESeq2.[Results] The analytic sample included 321 children with both PA and 16S rRNA sequencing data (mean age [SD] =10.2 [0.8] years; 54.2% male; 62.9% African American), where 189 (58.9%) met the PA recommendations. After adjusting for covariates, meeting the PA recommendations as well as minutes/day PA variables were not significantly associated with gut richness, evenness, or diversity (p ≥ 0.19). However, meeting the PA recommendations (weighted UniFrac R² = 0.014, p = 0.001) was significantly associated with distinct gut bacterial composition. These compositional differences were partly characterized by increased abundance of Megamonas and Anaerovorax as well as specific Christensenellaceae_R-7_group taxa in children with higher PA.[Conclusion] Children who met the recommendations of PA had altered gut microbiota compositions. Whether this translates to a reduced risk of obesity or associated metabolic diseases is still unclear.     

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.     

Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti‐bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat‐me” signals such as galectin‐8 and ubiquitin that label bacteria as autophagy cargo. Anti‐bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti‐bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella‐associated “eat‐me” signals, including host‐derived glycans and K48‐ and K63‐linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.     

Codon usage bias and the evolution of influenza A viruses. Codon Usage Biases of Influenza Virus

Background  

The influenza A virus is an important infectious cause of morbidity and mortality in humans and was responsible for 3 pandemics in the 20^th century. As the replication of the influenza virus is based on its host's machinery, codon usage of its viral genes might be subject to host selection pressures, especially after interspecies transmission. A better understanding of viral evolution and host adaptive responses might help control this disease.     

CD49d overexpression and T cell autoimmunity

D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.