Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P₁) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P₁ internalization is necessary for this effect. We characterize a knockin mouse (S1p1r^S5A/S5A) in which the C-terminal serine-rich S1P₁ motif, which is important for S1P₁ internalization but dispensable for S1P₁ signaling, is mutated. T cells expressing the mutant S1P₁ showed delayed S1P₁ internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P₁ agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P₁ expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P₁ on T cells is a primary determinant of lymphocyte egress kinetics in vivo.Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator that signals via five G protein–coupled receptors (GPCRs), regulates vascular maturation, permeability, and angiogenesis (Hla, 2004; Cyster, 2005). Recently, interest in the roles of S1P and its receptors in the immune system has been prompted in part by the identification of the immunomodulator FTY720 (Brinkmann et al., 2002; Mandala et al., 2002; Chiba, 2005), which upon phosphorylation by Sphk2 to FTY720-P (Sanchez et al., 2003; Zemann et al., 2006) acts as a strong agonist for four out of five S1P receptors (Brinkmann et al., 2004). FTY720 induces profound lymphopenia by inhibiting the egress of lymphocytes from the thymus, peripheral lymph nodes, and Peyer’s patches (Chiba, 2005). Indeed, it is now appreciated that S1P signaling modulates the trafficking of not only naive and central memory T cells, but also B cells, dendritic cells, NK cells, osteoclasts, and hematopoietic progenitor cells (Allende and Proia, 2002; Kabashima et al., 2006; Massberg et al., 2007; Schwab and Cyster, 2007; Walzer et al., 2007; Ledgerwood et al., 2008; Rivera et al., 2008; Sebzda et al., 2008; Ishii et al., 2009). These studies suggest that S1P regulates hematopoietic and immune cell trafficking under homeostatic and disease conditions; however, it is unclear precisely how S1P receptor signaling modulates cellular responses to egress cues.The mechanism of how S1P regulates T cell trafficking has been intensively investigated; T cell–specific deletion of S1p1r or hematopoietic reconstitution using S1p1r^−/− fetal liver cells resulted in profound lymphopenia, suggesting that the T cell–intrinsic S1P receptor 1 (S1P₁) is essential for their egress from the thymus and secondary lymph nodes (Allende et al., 2004; Matloubian et al., 2004). This observation, coupled with the finding that FTY720-P induces the loss of cell-surface S1P₁ from lymphocytes in an irreversible manner (Gräler and Goetzl, 2004; Matloubian et al., 2004), suggests that functional antagonism of S1P₁ in the lymphocyte compartment is essential for the inhibition of T cell egress.However, other studies have led to the proposal of an alternative mechanism by which S1P₁ regulates lymphocyte egress. Immunofluorescence microscopy demonstrated high expression levels of S1P₁ in endothelial cells, whereas staining of lymphocytes was weaker (Singer et al., 2005; Sinha et al., 2009). Moreover, administration of SEW2971, a selective S1P₁ agonist, does not induce irreversible receptor loss from the cell surface but causes significant lymphopenia in vivo (Jo et al., 2005). Two-photon microscopy of explanted lymph nodes containing labeled lymphocytes suggested that S1P₁ agonists may modulate barrier function and closure of vascular portals in the medulla, through which T cells egress into efferent lymphatics (Wei et al., 2005). Thus, this alternative proposal favors endothelial cells as the primary target cell type for S1P₁ agonists to inhibit lymphocyte egress (Rosen et al., 2008).Close interactions between immune and vascular cells may underlie the ability of S1P₁ to promote lymphocyte egress. In lymph node cortical sinuses, egress of T and B cells required S1P₁-dependent transendothelial traverse (Grigorova et al., 2009; Sinha et al., 2009). Indeed, competing chemotactic signaling between the egress-promoting S1P–S1P₁ system and the retention-promoting CXCL21–CCR7 chemokine receptor system of T cells appears to determine the rate and extent of their egress from secondary lymphoid organs (Pham et al., 2008). Whether S1P₁ signaling in lymphocytes, endothelial compartments, or both is important in the process of egress is not known.S1P₁ is a type I GPCR that is rapidly phosphorylated upon agonist stimulation. Although several protein kinases are involved in the phosphorylation of S1P₁ (Lee et al., 2001), phosphorylation at the C-terminal domain is particularly relevant to receptor desensitization and internalization (Hla, 2001). Because FTY720-P is degraded less efficiently than S1P by S1P lyase and S1P phosphatases (Bandhuvula et al., 2005; Mechtcheriakova et al., 2007; Yamanaka et al., 2008), its ligation likely induces sustained receptor activation kinetics. Presumably, this underlies the FTY720-P–induced irreversible internalization and proteosomal degradation of S1P₁ and resultant lymphopenia (Oo et al., 2007). The GRK-2 enzyme is capable of phosphorylating the serine-rich motif in the C-terminal tail of S1P₁ (Watterson et al., 2002), and we recently demonstrated that mutation of the five serines in the C terminus of S1P₁ to nonphosphorylatable alanines inhibited S1P- and FTY720-P–induced receptor internalization in transfected HEK293 cells (Oo et al., 2007). Although previous studies of GPCR signaling and chemotaxis have provided some insights into the role of internalization in these processes, the results appear to be receptor specific. For example, a CXCR4 superagonist induced greater chemotaxis than the native ligand stromal cell–derived factor–1α (SDF-1α) with no perceptible receptor internalization (Sachpatzidis et al., 2003). Conversely, mutations in the C terminus of CXCR2 resulted in defective receptor internalization concomitant with impaired chemotaxis (Sachpatzidis et al., 2003). In the case of S1P₁, it is unknown whether internalization is required for lymphocyte egress and recirculation.To address the role of S1P₁ internalization in the control of lymphocyte egress during homeostasis and FTY720 treatment, we developed a mouse model in which WT S1P₁ is replaced by the internalization-deficient mutant (S5A-S1P₁). We show that although T cell trafficking under homeostasis is unaltered, S1p1r^S5A/S5A mice display kinetic resistance to lymphopenia induced by the S1P₁ modulator (FTY720-P) or disruption of the S1P gradient. Adoptive transfer of S1p1r^WT/WT and S1p1r^S5A/S5A lymphocytes and S1P₁ surface staining of lymph node endothelial cells demonstrate that the T cell S1P₁, and not endothelial cell S1P₁ expression, regulates the rate of lymphocyte egress in vivo. These data support a T cell–intrinsic model of S1P₁ signaling in egress kinetics wherein the internalization of S1P₁ is a crucial modulator of the cues for T cell migration.     

Is fecundability associated with month of birth? An analysis of 19th and early 20th century family reconstitution data from The Netherlands

The relationship between fecundability and month of birth was investigated in a cohort of 1526 women who married between 1802 and 1929, using only women whose first marriage occurred before the age of 35 years. On the basis of their time to pregnancy (TTP, calculated as time between wedding and first birth minus gestational length), women were categorized into two groups: fecunds (TTP up to 12 months or prenuptial conceptions, n = 1348) and subfecunds (TTP >18 months, n = 118). By use of logistic regression, cosinor functions with a period of 1 year or 6 months and variable shift and amplitude were fitted through the monthly odds of subfecunds versus fecunds. The best fitting curve was unimodal, with a zenith in September (P = 0.13 for H0: no differences). Exclusion of childless women (n = 36, minimum follow-up 5 years) from the subfecunds led to a similar curve (P < 0.01), while childless women, as compared with fecunds, showed a birth distribution that was best represented with a bimodal curve with zeniths in January and July (P = 0.06). This study provides evidence for the existence of differences in fecundability by month of birth. The cause of this relationship is unclear, but may lie in a melatonin-dependent circannual variability of the quality of the oocyte.      

Mutation of the protein tyrosine kinase consensus site in the herpes simplex virus 1 alpha22 gene alters ICP22 posttranslational modification

We previously reported that at least eight HSV-1 and five HSV-2 proteins were tyrosine phosphorylated in infected human and mouse cells and the first phosphotyrosine-modified gene product identified was the ICP22 regulatory protein (Blaho, J. A., Zong, C. S., and Mortimer, K. A., 1997, J. Virol. 71, 9828-9832). All electrophoretic forms of ICP22 are tyrosine phosphorylated with the exception of the fastest migrating (unmodified) isoform. We now report the following. (i) ICP22 that reacted with a specific anti-phosphotyrosine antibody contained a significant amount of phosphotyrosine based on phospho-amino acid analysis. These results validate the discovery of ICP22 tyrosine phosphorylation. (ii) Wild-type ICP22 extracted from infected HEp-2 cells migrated as at least seven isoforms, termed ICP22a-g, in denaturing gels. (iii) The primary structure of ICP22 possesses a sequence that is homologous to protein tyrosine kinase recognition sites. A virus, termed RF141, was generated in which ICP22 tyrosine(193) in the kinase target site was mutated to an alanine. (iv) Biochemical analyses of infected HEp-2 and primary HFF cells indicated that the distributions of ICP22 isoforms differed between RF141 and control HSV-1(F). (v) The accumulations of representative viral polypeptides in RF141-infected HEp-2 cells appeared similar to wild-type virus. (vi) RF141 had reduced efficiencies of plating in HFF cells compared to control Vero cells. These differences increased as the multiplicity of infection decreased. Based on these results, we conclude (vii) that ICP22 tyrosine(193) is required for optimal posttranslational modification of the protein in HSV-1 infected human epithelial HEp-2 and primary human fibroblast cells.     

Immunohistochemical characteristics of diffuse sclerosing variant of papillary carcinoma: comparison with conventional papillary carcinoma

Koo JS, Shin E, Hong SW. Immunohistochemical characteristics of diffuse sclerosing variant of papillary carcinoma: comparison with conventional papillary carcinoma. APMIS 2010; 118: 744–52. Diffuse sclerosing variant of papillary carcinoma (DSVPC) is a rare variant of papillary thyroid carcinoma (PTC). It shows different clinicopathologic features to the conventional PTC, but the immunohistochemical characteristics of DSVPC are yet to be more clearly defined. The purpose of this study was to investigate the immunohistochemical features of DSVPC, which are different from those of PTC. Tissue microarray was constructed from the paraffin‐embedded tissue of 49 DSVPC and 50 conventional PTC samples. Immunohistochemical stains for p63, p53, galectin‐3, cytokeratin 19, β‐catenin, Bcl‐2, EMA, E‐cadherin, CD15, and CD56 were performed on each tissue microarray. Immunohistochemical stain for p63 was negative in all conventional PTCs, but 14 (28.6%) cases of DSVPC showed p63 expression (p = 0.000). p53 was expressed in 38 (76.0%) cases of conventional PTC and 21 (42.9%) cases of DSVPC (p = 0.001). Galectin‐3 was expressed in all 50 cases of conventional PTC, but eight (16.3%) cases of DSVPC did not express galectin‐3 (p = 0.003). EMA was expressed more in DSVPC (40.8%) than in conventional PTC (20.0%, p = 0.024). In univariate analyses, Bcl‐2 positivity (p = 0.016) and EMA negativity (p = 0.036) in DSVPC were associated with shorter time interval to tumor recurrence, but there was no significance for the two in multivariate analyses. DSVPC, a rare variant of PTC, has different immunohistochemical features from the conventional PTC, showing higher expression rate of p63 and lower expression rate of p53. It also shows galectin‐3 negativity and EMA positivity.     

Behavioural Effects of the Methanolic Root Bark Extract of Securinega Virosa in Rodents

Securinega virosa is used traditionally as sedative in children and in mental illnesses. In this study, the behavioral effects of methanolic root bark extract of S. virosa were investigated in mice. The results revealed that the extract significantly (P<0.05) and dose-dependently reduced the onset and prolonged the duration of sleep. The extract significantly (P<0.05) decreased exploratory activity and reduced the rate of apomorphine-induced stereotyped climbing at the doses tested (6.25–25mg/kg). It also produced a significant and dose-dependent motor coordination deficit in mice at the doses tested (P<0.01). The intraperitoneal median lethal dose in mice was 774.6mg/kg while the preliminary phytochemical screening revealed the presence of alkaloids, tannins, saponins and flavonoids. These results suggest that methanolic root bark extract of S. virosa contains biologically active principles that are sedative in nature and lend pharmacological credence to the ethnomedical use of the plant.     

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Anxiety during pregnancy and fetal attachment after in-vitro fertilization conception

The aim of this study was to compare 70 couples who had conceived by in- vitro fertilization (IVF) with 63 matched controls for the prevalence of anxiety and quality of attachment to the baby during pregnancy. Results for mothers showed no group differences using a global measure of anxiety, the Spielberger State-Trait Anxiety Inventory. However, pregnancy-specific measures revealed significantly higher levels of anxiety in IVF mothers about the survival and normality of their unborn babies, about damage to their babies during childbirth and about separating from their babies after birth. When IVF mothers were differentiated according to the number of treatment cycles, more differences in anxiety level were revealed, with most increases occurring in mothers who had experienced two or more treatment cycles. IVF fathers did not differ from controls on the global anxiety measure. No data on pregnancy-specific anxiety were available for fathers. Neither IVF mothers nor IVF fathers differed from controls on measures of attachment to the baby during pregnancy. Results are discussed in the context of the need for researchers to employ differentiated and issue-specific measures to identify concerns that may be unique to IVF couples. Clinical implications regarding the need for psychological support during pregnancy are also discussed.