Paracetamol poisoning induces acute liver injury in rats: inhibition of miR-155/cd45 axis-mediated antioxidant depletion and hepatotoxicity using quercetin and resveratrol / La intoxicación por paracetamol induce daño hepático agudo en ratas: inhibición de la depleción de antioxidantes mediado por el eje miR-155/cd45 y hepatotoxicidad usando quercetina y resveratrol

SUMMARY: Ingestion of an overdose of paracetamol (also called acetaminophen, or APAP) induces hepatotoxicity that can lead to liver failure. The link between the pro-inflammatory microRNA-155 (miR-155) and leukocyte infiltration (CD45) in APAP- antioxidant depletion and liver toxicity with and without the natural polyphenolic compounds, quercetin (QUR) plus resveratrol (RES) has not been previously studied. Therefore, acute hepatic injury was induced in rats by 2 g/kg APAP (single dose, orally) and another group started QUR (50 mg/kg) plus RES (30 mg/kg) treatment one week prior to APAP ingestion. Animals were culled 24 hours post the paracetamol treatment. APAP overdose induced hepatic and blood levels of miR-155 expression, CD45 (leukocyte common antigen) immunostaining, degenerated hepatocytes, and hepatic injury enzymes; alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were markedly decreased by QUR+RES. Whereas, APAP intoxication ameliorated liver tissue levels of the antioxidants, glutathione peroxidase and superoxide dismutase that were augmented by QUR+RES. Moreover, a significant (p<0.05) correlation between miR-155/CD45 axis and liver tissue injury was observed. These findings show that paracetamol intoxication augments miR- 155/CD45 axis-mediated modulation of antioxidants and liver injury in rats, and is protected by QUR+RES.

RESUMEN: La ingestión de una sobredosis de paracetamol (también llamado acetaminofeno o APAP) induce hepatotoxicidad que puede provocar insuficiencia hepática. El vínculo entre el microARN-155 proinflamatorio (miR-155) y la infiltración de leucocitos (CD45) en el agotamiento de APAP- antioxidante y la toxicidad hepática con y sin los compuestos polifenólicos naturales, quercetina (QUR) más resveratrol (RES) no ha sido previamente investigado. En este estudio, se indujo daño hepático agudo en ratas con 2 g/kg de APAP (dosis única, por vía oral) y otro grupo comenzó el tratamiento con QUR (50 mg/ kg) más RES (30 mg/kg) una semana antes de la ingestión de APAP. Los animales se sacrificaron 24 horas después del tratamiento con paracetamol. La sobredosis de APAP indujo niveles hepáticos y sanguíneos de expresión de miR-155, inmunotinción de CD45 (antígeno leucocitario común), degeneración de los hepatocitos y daño hepático enzimático; alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST), disminuyeron notablemente con QUR+RES. Mientras que la intoxicación con APAP mejoró los niveles de antioxidantes, glutatión peroxidasa y superóxido dismutasa en el tejido hepático los que aumentaron con QUR+RES. Además, se observó una correlación significativa (p<0,05) entre el eje miR-155/CD45 y la lesión del tejido hepático. Estos hallazgos muestran que la intoxicación por paracetamol aumenta la modulación mediada por el eje miR-155/CD45 de los antioxidantes y la lesión hepática en ratas, y está protegida por QUR+RES.

A feasibility study of combined epigenetic and vaccine therapy in advanced colorectal cancer with pharmacodynamic endpoint.

Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3-4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.

Deltamethrin-Induced Immunotoxicity and its Protection by Quercetin: An Experimental Study.

BACKGROUND: Deltamethrin (DLM) is a type 2 pyrethroid insecticide used in agriculture and home to control pests. However, emerging reports have indicated the immunotoxicity of DLM. OBJECTIVE: Thus, in the current investigation, we have checked the immune-protective role of quercetin in DLM-induced immunotoxicity by using in silico and in vitro techniques. RESULTS: In silico results have shown good interaction of quercetin towards immune cell receptors (T & B cell receptors). The findings of in vitro studies indicated the decrease in oxidative stress which is elevated by DLM in concentration & time-dependent manner. The increased caspases-3 activity was decreased by treatment of quercetin. The apoptosis induced by DLM in thymus and spleen was suppressed only at higher concentration (50µg/ml) of quercetin. Finally, the phenotypic changes due to DLM were restored by quercetin. CONCLUSION: Quercetin has strong binding affinity towards CD4, CD8 and CD28, CD45 receptors and protects the thymocytes and splenocytes against DLM-induced apoptotic signaling pathways.

Mechanisms involved in CD4 cell gains in HIV-infected patients switched to raltegravir.

BACKGROUND: CD4 gains in HIV patients on HAART result from release of T cells recently migrated from the thymus, redistribution from lymphoid tissues, proliferation in the periphery and/or reduced apoptosis. The relative contribution of each mechanism in CD4 restoration in patients with suppressed viremia switching antiretrovirals is unclear. METHODS: HIV patients with undetectable viremia on HAART were identified at our clinic. A subset switched to raltegravir was compared with another group that kept therapy unmodified. Naive and memory CD4 T-cells were measured by flow cytometry using CD45RA and CD27, respectively. Activation was examined using CD38 and recent thymic emigrants using CD31. Apoptosis was analyzed measuring soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL). RESULTS: Thirty-seven patients were examined, 19 switched to raltegravir and 18 controls, after a median of 26 months of suppressed viremia. At 6 months, mean CD4 cell counts significantly increased in raltegravir patients from 322 to 448 cells/µl (P = 0.026) but not in controls (from 312 to 330 cells/µl; P  = 0.813). No significant changes were recognized in activation or CD31 expression in any group. In raltegravir patients, however, the proportion of naive CD4 T cells significantly increased (P = 0.014) as well as CD38 expression in these cells (P = 0.036). A positive correlation was found between CD38 and CD31 expression in naive CD4 T cells (R  = 0.51, P < 0.001). TRAIL and FasL did not decline significantly in any group. CONCLUSION: HIV patients with prolonged undetectable viremia on HAART experience more pronounced CD4 gains after raltegravir switching than keeping the same regimen. An increased production of naive CD4 T cells largely explains this effect.

Valproic acid induces microglial dysfunction, not apoptosis, in human glial cultures.

Valproic acid (VPA) is widely used for the treatment of mood disorders and epilepsy, but its mechanism of action is unclear. In vivo and in vitro studies using rodent models have demonstrated that VPA has both neuroprotective and neurotrophic effects. These beneficial effects are, in part, through modulation of glial cell function. Recently, we and others have shown that VPA selectively induces caspase-3 mediated apoptosis in rodent microglial cells. However, the effect of VPA on human microglia has not been tested. In this study, using microglia derived from adult human brains, we demonstrate that VPA does not induce microglial apoptosis as determined by the absence of caspase-3 cleavage. However, VPA does partially decrease the expression of the microglial markers PU.1 and CD45, as well as dramatically reducing microglial phagocytosis. Due to the many roles of microglia in the brain, these VPA-induced alterations in microglial phenotype could potentially have major effects on physiological and pathological actions of these cells.

Anti-CTLA-4 antibody therapy: immune monitoring during clinical development of a novel immunotherapy.

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T-cell activation. Antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including antitumor immunity. Two CTLA-4-blocking antibodies are presently under clinical investigation: ipilimumab and tremelimumab. CTLA-4 blockade has shown promise in treatment of patients with metastatic melanoma, with a recently completed randomized, double-blind phase III trial demonstrating a benefit in overall survival (OS) in the treated population. However, this approach appears to benefit only a subset of patients. Understanding the mechanism(s) of action of CTLA-4 blockade and identifying prognostic immunologic correlates of clinical endpoints to monitor are presently areas of intense investigation. Several immunologic endpoints have been proposed to correlate with clinical activity. This review will focus on the endpoints of immune monitoring described in studies to date and discuss future areas of additional work needed.

Altered B cell development and anergy in the absence of Foxp3.

The importance of regulatory T cells in immune tolerance is illustrated by the human immune dysregulatory disorder IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), caused by a lack of regulatory T cells due to decreased or absent expression of Foxp3. Although the majority of work on regulatory T cells has focused on their ability to suppress T cell responses, the development of significant autoantibody titers in patients with IPEX suggests that regulatory T cells also contribute to the suppression of autoreactive B cells. Using a murine model, deficient in the expression of Foxp3, we show that B cell development is significantly altered in the absence of regulatory T cells. Furthermore, we identify a loss of B cell anergy as a likely mechanism to explain the production of autoantibodies that occurs in the absence of regulatory T cells. Our results suggest that regulatory T cells, by either direct or indirect mechanisms, modulate B cell development and anergy.

Flow cytometric analysis of age-related changes in intestine intraepithelial lymphocyte subsets and their functional preservation after feeding mice on spirulina.

We investigated age-related changes in intestinal intraepithelial lymphocyte (IEL) subsets in mice by flow cytometric analysis and their functional preservation as affected by feeding Spirulina, a cyanobacterium that is known to possess various therapeutic effects, including immune modulation activity. The number of cells possessing the leukocyte-common antigen CD45(+) cells in mice (43 weeks old) of the aged group, used as a representative marker for IELs, was significantly lower than that of adult mice (5 weeks old). Either the proportion or the number of CD45(+)CD8(+) cells of the aged mice was significantly lower than that of adult mice, corresponding to previous reports. Proportions and numbers of CD4(+)CD8(+) cells in aged mice, on the other hand, were higher than those in adult mice. Increased or decreased levels of the cell surface antigens observed in the aged mice tended to be restored in aged mice fed Spirulina (aged-SP group), which ingested a hot water extract of Spirulina (SpHW). In fact, the proportions of CD45(+)CD8(+) cells and CD45(+)TCRgammadelta(+) cells in the aged-SP group significantly increased in comparison to the control aged group, which ingested ordinary chow and water ad libitum. These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against infectious agents through retaining the number of certain IELs. Changes in the number of other IEL subsets and blood cells are also discussed.

Influence of antiretroviral therapy on programmed death-1 (CD279) expression on T cells in lymph nodes of human immunodeficiency virus-infected individuals.

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

Differential effects of the dopamine neurotoxin MPTP in animals with a partial deletion of the GDNF receptor, GFR alpha1, gene.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor beta (TGFbeta) superfamily, is a potent neurotrophic protein promoting the survival and maintenance of dopaminergic (DA) neurons in the substantia nigra during development and adulthood. DA neurons that project to the striatum in the nigrostriatal pathway express GDNF receptors, GFR alpha1. The purpose of this study was to determine whether these neurons are especially sensitive to neurotoxic insults. Therefore, we examined effects of the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on locomotion and DA neurons in 26-month-old male GFR alpha1 heterozygous (GFR alpha1(+/-)) mice compared to aged-matched wild-type (WT) littermates. MPTP gave rise to increased locomotion, regardless of genotype, while GFR alpha1(+/-) mice treated with saline exhibited lower spontaneous locomotion, compared to WT mice. Moreover, GFR alpha1(+/-) saline mice had fewer TH-positive neurons, greater expression of inflammatory markers (CD45 immunostaining and phosphorylated p38 MAPK) in the nigra, and reduced striatal TH staining. MPTP exacerbated these effects, with the lowest density of striatal TH and highest density of nigral CD45 and phospho-p38 MAPK immunoreactivity observed in GFR alpha1(+/-) mice. The findings point to increased sensitivity of the DAergic system with age and neurotoxic exposure as a result of a genetic reduction of GFR alpha1.

CD45RB is a novel molecular therapeutic target to inhibit Abeta peptide-induced microglial MAPK activation.

BACKGROUND: Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer's disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed beta-amyloid (Abeta) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation. METHODOLOGY AND RESULTS: In this study, CD45RB modulation of Abeta peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with "aged" FITC-Abeta(1-42) and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Abeta(1-42) peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-alpha and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Abeta(1-42) peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Abeta(1-42) peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-alpha and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Abeta(1-42) peptide, inhibits co-localization of microglial MHC class II and Abeta peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells. CONCLUSION: In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Abeta phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Abeta lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.

Use of transcriptional synergy to augment sensitivity of a splicing reporter assay.

A primary limitation in the development and use of screens to identify factors that regulate mammalian pre-mRNA splicing has been the development of sensitive reporter assays. Alternative splicing typically involves relatively small (< 10-fold) changes in isoform ratios. Therefore, reporter constructs designed to allow direct analysis of isoform expression historically have at most a 10-fold window of discrimination between a positive signal and background. Here we describe the design and application of a reporter cell line that makes use of the phenomenon of transcriptional synergy to amplify the detection of changes in splicing, such that a three- to five-fold change in splicing pattern is observed as a 30- to 50-fold change in GFP expression. Using this cell line we have identified two small molecules, from a library of approximately 300 synthetic compounds, that can induce partial repression of a variable exon from the CD45 gene. We propose that the concept of transcription-based amplification of signal will allow the development of true high-throughput screening approaches to identify effectors of mammalian alternative splicing.

Etanercept treatment in the endotoxin-induced uveitis of rats.

This study was conducted to investigate therapeutic value of a soluble tumor necrosis factor-alpha (TNF-alpha) receptor, etanercept, in a rat model of endotoxin-induced uveitis (EIU). Forty-two inbred male Lewis rats were divided into seven equal groups. 200 microg of Escherichia coli 055:B55 lipopolysaccharide (LPS) was injected in one hind footpad of the Groups 2, 3, 4, 5, 6, and 7 rats. Group 5, 6, and 7 rats also received subcutaneous etanercept 24 hr prior to LPS injection at a dose of 0.4 mg kg(-1). Group 1 rats were used as controls. Eight, 24, and 48 hr after treatment clinical uveitis scores (miosis, iris hyperemia, and hypopyon) were assessed by a masked observer and the rats were euthanized. Neutrophil leukocytes, CD8+, CD4+, and CD45RO+ cells in the anterior uveal tissue were counted either after hematoxylin-eosin or monoclonal antibody staining. TNF-alpha levels were also measured in the aqueous humor samples by an ELISA method. Etanercept treatment significantly improved clinical uveitis scores at all examination points compared to the LPS injected animals. The improvement was almost complete expect for the miosis score, since no significant difference was detected between the controls and LPS + Etanercept treated animals at all examination points. Cell counts were also at significantly lower levels in LPS + Etanercept treated animals at all examination points, except for CD8+ and CD45RO+ cell counts at 24 hr examination point. There was no significant difference between the controls and LPS + Etanercept treated animals at all examination points as with CD4+ and CD45RO+ cell counts at 48 hr. Our data showed that etanercept had a definite effect on the treatment of EIU. Further studies should clarify its efficacy on clinical uveitis conditions.

Decrease of interleukin-10-producing T cells in the peripheral blood of severe unstable atopic asthmatics.

BACKGROUND: Although IL-10 is known as an immunoregulatory cytokine produced by various cells including T cells, its basic profile in atopic asthma remains uncertain. OBJECTIVE: The profiles of IL-10 production in circulating CD4+ T cells of atopic asthmatics were investigated with respect to clinical severity. METHODS: Forty atopic asthmatics were divided into three groups: mild, and severe but stable and severe unstable asthmatics. Eosinophils were counted in the peripheral blood and sputum, and exhaled nitric oxide was assessed. PBMCs were stimulated with or without anti-CD3 and anti-CD28 antibodies and then processed for detecting IL-10-producing CD4+ cells using flow cytometry. RESULTS: There was no difference in the eosinophil count in blood or sputum and in nitric oxide level among the three groups. IL-10-producing CD4+ cells were mainly detected in a CD45RO+ memory population. The frequency of IL-10-producing cells after stimulation was significantly lower in the severe unstable group compared to the mild group. In addition, the frequency of IL-10-producing cells in the severe unstable group was significantly lower than that in the severe stable group despite the fact that both groups received similar treatments with high-dose inhaled corticosteroids. The IL-10 production of CD4+CD45RO+ cells in response to dexamethasone did not differ among the three groups. CONCLUSIONS: IL-10-producing CD4+CD45RO+ cells in the peripheral blood are decreased in severe unstable asthmatics, which is not explained by the effect of high-dose inhaled corticosteroid medication.

Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death.

The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.

Age-related immune reconstitution during highly active antiretroviral therapy in human immunodeficiency virus type 1-infected children.

OBJECTIVES: To evaluate the immune reconstitution in HIV-1-infected children in whom highly active antiretroviral therapy (HAART) controlled viral replication and to assess the existence of a relation between the magnitude of this restoration and age. METHODS: All HIV-1-infected children in whom a new HAART decreased plasma viral load below 400 copies/ml after 3 months of therapy were prospectively enrolled in a study of their immune reconstitution. Viral load, lymphocyte phenotyping, determination of CD4+ and CD8+ T cell receptor repertoires and proliferative responses to mitogens and recall antigens were assessed every 3 months during 1 year. RESULTS: Nineteen children were evaluated. Naive and memory CD4+ percentages were already significantly increased after 3 months of HAART. In contrast to memory CD4+ percentages, naive CD4+ percentages continued to rise until 12 months. Age at baseline was inversely correlated with the magnitude of the rise in naive CD4+ cells after 3, 6 and 9 months of therapy but not after 12 months. Although memory and activated CD8+ cells were already decreasing after 3 months, abnormalities of the CD8 T cell receptor repertoire and activation of CD8+ cells persisted at 1 year. HAART increased the response to mitogens as early as 3 months after starting therapy. CONCLUSIONS: In children the recovery of naive CD4+ cells occurs more rapidly if treatment is started at a younger age, but after 1 year of viral replication control, patients of all ages have achieved the same level of restoration. Markers of chronic activation in CD8+ cells persist after 1 year of HAART.

Mechanisms involved in the differential bone marrow homing of CD45 subsets in 5T murine models of myeloma.

Multiple myeloma (MM) is an incurable plasma cell cancer, localized in the bone marrow (BM). The mechanisms used by these cells to (re-)enter this organ remain largely unknown. Recently, we reported that both CD45+ and CD45- myeloma cells home to the BM and induce myeloma disease. In this work, we investigated the underlying mechanisms involved in the homing of CD45+ and CD45- myeloma cells in the experimental 5T2MM and 5T33MM murine models. In vivo tracing of flow cytometric sorted and radioactively labeled CD45 subsets revealed a reduced homing of the CD45- 5TMM cells to the BM as compared to the CD45+ 5TMM cells. Migration assays demonstrated an impaired chemotaxis towards BM endothelial cell conditioned medium, BM stromal cell conditioned medium and towards the basement membrane component laminin-1 of the CD45- 5TMM cells compared to the CD45+ subset. Matrix metalloproteinase-9 (MMP-9) and urokinase type plasminogen activator (uPA) are key extracellular matrix proteases involved in the invasion of cancer cells. Inhibitor and antibody blocking experiments demonstrated the involvement of both in the invasion of the 5TMM cells. CD45- 5TMM cells had a low secretion of MMP-9 and (for the non-aggressive line 5T2MM only) a low cell surface expression of uPA receptor, as revealed by gelatin zymography and flow cytometric analysis, respectively. Accordingly, the synthetic basement membrane invasive capacity of the CD45- 5TMM subpopulations was also impaired. Our results indicate that CD45+ and CD45- 5T myeloma cells have a differential BM homing attributable to differential migratory and invasive capacities.

Efficient total synthesis of pulchellalactam, a CD45 protein tyrosine phosphatase inhibitor.

A new approach to a CD45 protein tyrosine phosphatase inhibitor, pulchellalactam, is described. The key step of the sequence involves addition and elimination of an enolic lactam in a single step and 70% yield, employing an organocuprate reagent. The resulting alpha,beta-unsaturated lactam could be condensed with isobutyraldehyde to produce Z-pulchellalactam or converted into siloxypyrrole, which was subjected to the BF(3) x Et(2)O-promoted coupling reaction with isobutyraldehyde to afford E-pulchellalactam after E1-cB elimination and TFA deprotection. This first total synthesis afforded Z-pulchellalactam in six steps and 32% overall yield from Boc-glycine. The same sequence of reactions could also be applied to the liquid- or solid-phase synthesis of trifunctionalized pulchellalactam derivatives.

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45.

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.