Fyn is involved in angiotensin II type 2 receptor-induced neurite outgrowth, but not in p42/p44mapk in NG108-15 cells.

In NG108-15 cells, activation of p42/p44(mapk) is essential for induction of neurite outgrowth by angiotensin II (Ang II) type 2 receptor (AT(2)). The aim was to verify whether Fyn, a member of the Src family kinases (SFK), is involved in neurite outgrowth induced by AT(2) activation. Preincubation of cells with PP1, a general inhibitor of the SKF, decreased activation of Rap1 and p42/p44(mapk) and abolished TrkA activation by Ang II or by the AT(2) agonist, CGP42112A. NG108-15 cells were transfected with a Fyn-WT and a Fyn-DN expressing vector. Fyn-WT was sufficient to induce neurite outgrowth, although transfection with Fyn-DN abolished neurite elongation. However, the Fyn-DN form failed to affect activation of TrkA, Rap1 or p42/p44(mapk) by Ang II. Thus, although SKF activity is required to achieve AT(2)-induced activation of TrkA, Rap1 and p42/p44(mapk), Fyn is essential for AT(2) receptor-induced neurite outgrowth, but not in AT(2) signaling leading to p42/p44(mapk) activation.

Engraftment of bone marrow from severe combined immunodeficient (SCID) mice reverses the reproductive deficits in natural killer cell-deficient tg epsilon 26 mice.

A large, transient population of natural killer (NK) cells appears in the murine uterine mesometrial triangle during pregnancy. Depletion of uterine (u) NK cells, recently achieved using gene-ablated and transgenic mice, results in pathology. Pregnancies from matings of homozygous NK and T cell-deficient tg epsilon 26 mice have <1% of normal uNK cell frequency, no development of an implantation site-associated metrial gland, and an edematous decidua with vascular pathology that includes abnormally high vessel walls/lumens ratios. Fetal loss of 64% occurs midgestation and placentae are small. None of these features are seen in pregnant T cell-deficient mice. To confirm the role of the NK cell deficiency in these reproductive deficits, transplantation of tg epsilon 26 females was undertaken using bone marrow from B and T cell-deficient scid/scid donors. Engrafted pregnant females have restoration of the uNK cell population, induced metrial gland differentiation, reduced anomalies in the decidua and decidual blood vessels, increased placental sizes, and restoration of fetal viability at all gestational days studied (days 10, 12, and 14). Thus, uNK cells appear to have critical functions in pregnancy that promote decidual health, the appropriate vascularization of implantation sites, and placental size.

Correction: Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNAs in graft-versus-host disease: a review of the latest data.

Detectable in biopsies and body fluids, the measurement of a single or panels of microRNAs have been reported to be quite sensitive and specific for the prediction, diagnosis, and prognosis of many diseases. Interest in the use of microRNAs as biomarkers and eventual therapeutic targets has increased exponentially in the last decade. However, in the field of graft-versus-host disease (GVHD), the discovery of their involvement in biological processes and their predictive value is only emerging. With 30-75% of patients developing GVHD following allogeneic hematopoietic cell transplant and the absence of routinely used predictive biomarkers, microRNAs are promising for early detection and follow-up of this condition. We aim to summarize the current knowledge on the involvement of these small RNAs in the pathophysiology of this disease. We also review studies investigating the potential of miRNAs as biomarkers for early detection, follow-up, and prognosis of GVHD.

Biogenesis of multilamellar bodies via autophagy.

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.

Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

Imatinib mesylate (IM) therapy has been shown to induce lower T cell counts in chronic myelogenous leukemia (CML) patients and an interference of IM with T cell receptor (TCR) signaling has been invoked to explain this observation. However, IL-7 and TCR signaling are both essential for lymphocyte survival. This study was undertaken to determine whether IM interferes with IL-7 or TCR signaling to explain lower T cell counts in patients. At diagnosis, CML patients have typically lower CD4+ counts in their blood, yet CD8+ counts are normal or even increased in some. Following the initiation of IM treatment, CD4+ counts were further diminished and CD8+ T lymphocytes were dramatically decreased. In vitro studies confirmed IM interference with TCR signaling through the inhibition of ERK phosphorylation and we showed a similar effect on IL-7 signaling and STAT5 phosphorylation (STAT5-p). Importantly however, using an in vivo mouse model, we demonstrated that IM impaired T cell survival through the inhibition of IL-7 and STAT5-p but not TCR signaling which remained unaffected during IM therapy. Thus, off-target inhibitory effects of IM on IL-7 and STAT5-p explain how T cell lymphopenia occurs in patients treated with IM.

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptide-treated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1-target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML.

Corneal and lenticular opacities in mentally retarded young adults treated with thioridazine and chlorpromazine.

Of 18 mentally retarded institutionalized subjects who had received long-term, high-dose treatment primarily with thioridazine or chlorpromazine, 2 developed definite corneal and lenticular opacities and 2 had equivocal ocular changes. In view of this finding, the authors suggest that alternative treatment, including different neuroleptics, be considered with mentally retarded institutionalized subjects; when that is not possible, subjects should be given periodic evaluation, including annual slit-lamp examination.

Immune competence involving the natural killer cell lineage promotes placental growth.

Very small placentae and absence of uterine natural killer (uNK) cells are amongst the reproductive deficits found in the natural killer (NK) cell and thymus-derived (T) cell immunodeficient mouse tgepsilon26. These defects can be reversed by grafting of adult tgepsilon26 females with bone marrow from T and B cell immunodeficient scid/scid donors. We report here that a second protocol, grafting of neonatal tgepsilon26 females with immunocompetent bone marrow pretreated with antibody to Thy-1, successfully established the uNK cell lineage and ameliorated the phenotype. Further, comparisons of mid-gestation (days 10-16) placental area measurements from tgepsilon26 and seven other immunodeficient strains to time-matched tissues from four strains of immunocompetent mice indicate that lymphocytes of the NK but not the T or B cell lineages are able to influence placental size during normal gestation and that this action is independent of interleukin 2. Area measurements of placentae produced in manipulated tgepsilon26 pregnancies (maternal bone marrow engraftment, outcrossing to immunocompetent males and reciprocal embryo transfers with an immunocompetent strain) suggest that NK cell competence is required in each of the maternal and fetal compartments to optimize placental growth.

Susceptibility of MHC class I expressing extravillous trophoblast cell lines to killing by natural killer cells.

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when cultured on laminin-coated plates. Very weak HLA-G expression was detectable on the cell surface under these conditions. However, strong cell surface expression of a classical MHC class I molecule (most likely HLA-C) was exhibited by these EVT cell lines when grown on laminin, as indicated by W6/32 FACS analysis (Ab recognizing pan MHC class I), and Western immunoblotting with HC10 (Ab recognizing HLA-B/C). When these EVT cells, cultured on laminin, were used as targets for peripheral blood natural killer (NK) cells in a standard chromium release assay, both HTR-8 and HT-116 cells were lysed by NK cells in a dose-dependent manner. The respective percentage specific lysis at an effector to target (E/T) ratio of 100 was 28+/-7, and 48+/-14. The choriocarcinoma cell lines JAR and JEG-3 which were respectively MHC class I negative and HLA-G positive were resistant to NK cell lysis. Thus, there was no clear relationship between the MHC class I expression and NK cell resistance or susceptibility among the EVT cell lines and choriocarcinoma cells. These findings raise the possibility that NK cells may take part in the surveillance of the invasive EVT cells during normal placentation.

Branhamella catarrhalis pathogenesis in SCID and SCID/beige mice. Brief report.

SCID and SCID/beige mice were used to study the pathogenesis of B. catarrhalis administered by intranasal, intraperitoneal or intravenous routes. Challenged adult animals did not appear overtly clinically ill. Similar symptoms were observed regardless of the challenge route, and pretreatment of mice with human transferrin did not enhance clinical virulence. Susceptibility to B. catarrhalis appeared to be age-dependent as some mice under one week of age died following challenge. Postmortem findings included circumscribed pale foci on the liver, splenomegaly and mineralization of the myocardium. Presence of lesions did not correlate with the assessment of clinical well being, and severity of the lesions was found to be challenge strain-dependent. Liver lesions and splenomegaly were not observed in animals challenged with heat-killed bacteria or placebo. SCID/beige mice were more affected than SCID mice both clinically and pathologically, suggesting that natural killer cell and polymorphonuclear cell functions may be important in resolving B. catarrhalis challenge.

Histological studies of gene-ablated mice support important functional roles for natural killer cells in the uterus during pregnancy.

Maternal lymphocytes having a large and granulated morphology accumulate at healthy implantation sites in normal mice. Insight into the functions of these cells has come from a previous study of two independent lines of mice deficient in natural killer (NK) cells. In pregnant Tg epsilon 26 mice, vascular pathology was found that led to the major complications of either fetal death or intrauterine growth retardation. In pregnant p56lck null x IL-2R beta null mice, extensive distension of the decidua was observed that separated the placenta from the myometrium and appeared to be interstitial edema. To strengthen assignment of uterine large granulated lymphocytes to the NK cell lineage and to understand which aspects of NK cell biology may be important for a uterine-based, pregnancy-associated subset, mid-gestation implantation sites from a new series of mice having gene deletions which alter NK cells (IL-2R gamma null, Stat4 null, IL-12 p40 null, beta 7 integrin null and Muc-1 null) have been examined histologically. The findings support the assignment of pregnancy-associated large granulated cells of mice to the NK cell lineage and suggest that the primary functions of these tissue-based NK cells are to support normal development of the decidua and/or its vasculature using pathways that involve IL-12 mediated signal transduction.

Glucose infusion partially attenuates glucose production and increases uptake during intense exercise.

Glucose infusion can prevent the increase in glucose production (Ra) and increase glucose uptake (Rd) during exercise of moderate intensity. We postulated that 1) because in postabsorptive intense exercise (>80% maximal O2 uptake) the eightfold increase in Ra may be mediated by catecholamines rather than by glucagon and insulin, exogenous glucose infusion would not prevent the Ra increment, and 2) such infusion would cause greater Rd. Fit young men were exercised at >85% maximal O2 uptake for 14 min in the postabsorptive state [controls (Con), n = 12] or at minute 210 of a 285-min glucose infusion. In seven subjects, the infusion was constant (CI; 4 mg . kg-1 . min-1), and in seven subjects it was varied (VI) to mimic the exercise Ra response in Con. Although glucose suppressed Ra to zero (with glycemia approximately 6 mM and insulin approximately 150 pM), an endogenous Ra response to exercise occurred, to peak increments two-thirds those in Con, in both CI and VI. Glucagon was unchanged, and very small increases in the glucagon-to-insulin ratio occurred in all three groups. Catecholamine responses were similar in all three groups, and correlation coefficients of Ra with plasma norepinephrine and epinephrine were significant in all. In all CI and VI, Rd at rest was 2x Con, increased earlier in exercise, and was higher for the 1 h of recovery with glucose infusion. Thus the Ra response was only partly attenuated, and the catecholamines are likely to be the regulators. This suggests that an acute endogenous Ra rise is possible even in the postprandial state. Furthermore, the fact that more circulating glucose is used by muscle during exercise and early recovery suggests that muscle glycogen is spared.

Pregnancy-associated uterine granulated metrial gland cells in mutant and transgenic mice.

PROBLEM: Granulated metrial gland (GMG) cells are pregnancy-specific uterine lymphocytes found in rodents. The lineage relationships of GMG cells are incompletely defined, although genetic and immunophenotyping studies suggest membership in the natural killer (NK) cell lineage. Pregnancy-specific functions have been postulated for GMG cells, but no successful depletion of these cells has been achieved that would permit assessment of any critical roles that might influence gestational outcome. METHOD: Routine histological methods for light microscopy were used to assess implantation sites from wild-type mice and mice of the following genotypes: tgE26, mi/mi, and p53 knockout. RESULTS: GMG cells are transient, histamine-negative cells found in the pregnant uteri of most mice. Pregnancies in the NK and T-cell-deficient tgE26 were characterized by infrequent GMG cells, very small placentae, and an elevated rate of fetal and perinatal mortality. In term placentae of mi/mi with dystocia. GMG cells were found in a new location along the plane of placental separation, and they appeared degranulated. A normal life-history was observed for GMG cells in p53 knockout mice. CONCLUSION: Pregnancies in mutant and transgenic mice indicate that GMG cells are natural killer cells and have critical roles in placental development and pregnancy success. The disappearance of GMG cells at term is independent of p53 gene expression.

Uterine natural killer cells: insights into lineage relationships and functions from studies of pregnancies in mutant and transgenic mice.

Mice expressing genetic alterations have been extremely valuable in providing insights into both the lineage relationships and functions of lymphohematopoietic cells. This approach has been applied to granulated lymphocytes that localize to implantation sites in the rodent uterus during pregnancy. Histological analyses of implantation sites collected from various mutant and transgenic mice over the course of gestation strongly support the conclusions that these granulated lymphocytes, previously named granulated metrial gland (GMG) cells, are natural killer (NK) cells and that they are essential for normal development of the placenta. Pregnancy-associated uterine NK (uNK) cells have limited lytic activity suggesting that their secretory products, particularly cytokines and matrix proteins, may be critical for normal placental maturation. This review will highlight information collected from pregnancies in mutant and transgenic mice that have contributed to the current understanding of functions of uNK cells during gestation.

Uterine natural killer cells do not require interleukin-2 for their differentiation or maturation.

PROBLEM: Natural Killer lymphocytes (NK cells) from the pregnant uterus and from other tissues in pregnant and nonpregnant mammals can be stimulated by interleukin-2 (IL-2) during culture to become Lymphokine Activated Killer (LAK) cells. The susceptibility of cultured trophoblast cells to lysis by LAK cells raises the enigma of why uterine (u) NK cells that are characterized by morphology and by surface phenotyping as "activated," and thus potentially damaging to the placenta, become localized to implantation sites during normal rodent gestation. METHOD: uNK cells migrating from explant cultures of the metrial gland were assessed for expression (mRNA and protein) of each chain of the IL-2 receptor (IL-2R). Implantation sites from transgenic mice lacking a functional IL-2 gene were examined histologically for the differentiation of mature, granulated uNK cells. RESULTS AND CONCLUSION: Early post-implantation, mRNA from migrating uNK cells contains transcripts for all three chains of the IL-2R. Only IL-2Rgamma was expressed at day 12 of gestation; expression of this gene was also lost by day 16. Loss of IL-2R transcription did not result in loss of protein expression; however, it did coincide with loss of uNK cell viability in vivo. Apparently normal differentiation of uNK cells occurred in IL-2(-/-) mice and in doubly mutant IL-2(-/-).beta2m(-/-) mice. Thus, despite uNK cell expression of the full IL-2R at day 8 of gestation, IL-2 is not required for the maturation of uNK cells to their fully granulated form or for normal placental development.

Absence of natural killer cells during murine pregnancy is associated with reproductive compromise in TgE26 mice.

Strategies of cell depletion were pursued to extend understanding of the functions of natural killer (NK) cell-like large granulated lymphocytes found in the rodent uterus during pregnancy. Repeated infusions of antibody to Ly-49G2, a surface marker thought to be expressed by the progenitor forms of these cells, removed Ly-49G2+ cells from the virgin but not the pregnant uterus. Large granulated uterine lymphocytes also differentiated during pregnancy in transgenic mice that carried a deletion in the IL-2 gene. This cell population was absent in two strains of mice, p56lck-/lck-.IL-2Rbeta-/IL-2Rbeta- and TgE26. Implantation sites in both of these strains had histopathological anomalies in the zone of decidualization. In TgE26 mice, a sudden onset of fetal loss began at Day 10 of gestation. Fetal death was associated with progressive changes in the maternal uterine arterioles, suggestive of localized arteriosclerosis associated with hypertension. TgE26 females carried immune-competent fetuses to term, apparently through preventive or compensatory mechanisms that may modify the uterine vasculature after the onset of vascular pathology. These studies are the first to suggest a vital role for large granulated lymphocytes in the promotion of fetal survival and pregnancy success.

Relapse after bone marrow transplantation: evidence for distinct immunological mechanisms between adult and paediatric populations.

Donor lymphocyte infusions are particularly effective for remission induction in malignant cells in patients who relapse after allogeneic progenitor cell transplantation (PCT) and who remain sensitive to the administration of unprimed donor T and/or natural killer (NK) cells present in donor lymphocyte infusions. To determine whether relapse after unmanipulated PCT could be ascribed to donor T and/or NK cell loss or tolerization, we evaluated the chimeric status of 81 patients with haematological malignancies who were receiving allogeneic unmanipulated PCT. The incidence of mixed chimaerism (MC) in unfractionated mononuclear leucocyte samples decreased rapidly after transplant, and was not detectable 4 months after PCT, even in patients who subsequently relapsed. The chimeric status of immune effector cell subsets was then evaluated in 15 patients at the time of relapse. All adults demonstrated complete donor haematopoiesis (CDH) for all cell lineages, whereas T- and NK-cell MC was only found in patients younger than age 13 years (P = 0.004). MC was not found in T nor NK cells of a control group consisting of age-matched paediatric patients in remission after allogeneic PCT. Thus, in adults, T and NK cell MC disappears early after unmanipulated allogeneic PCT and is absent at the time of relapse. However, the identification of donor T and NK cell loss in the paediatric relapsed but not remission patients suggests a distinct mechanism of relapse.

Strong association of autoantibodies to human nuclear lamin B1 with lupus anticoagulant antibodies in systemic lupus erythematosus.

OBJECTIVE: To determine the frequency and clinical significance of high titers of IgG autoantibodies to nuclear lamin B1 in a large number of unselected and well-characterized systemic lupus erythematosus (SLE) patients, disease controls, and normal healthy controls. METHODS: A cross-sectional study of anti-lamin B1 autoantibodies, as measured by enzyme-linked immunosorbent assay using human recombinant lamin B1 autoantigen, was performed on serum samples obtained at first evaluation of 238 consecutive French Canadian adults: 61 healthy control subjects, 20 patients with osteoarthritis, 22 with ankylosing spondylitis, 11 with autoimmune hepatitis, 30 with rheumatoid arthritis, and 94 with SLE. SLE patients were studied for 57 disease manifestations. A case-control study was performed to analyze the relationship between anti-lamin B1 status and thrombotic manifestations between SLE onset and last followup. RESULTS: High titers of anti-lamin B1 were strikingly restricted to a subset of 8 SLE patients (8.5%). The mean anti-lamin B1 titer was higher in this subset than in the other SLE patients or any control group (P<0.001). By univariate analysis and stepwise multiple logistic regression, the most striking association of anti-lamin B1 was with lupus anticoagulant (LAC) antibodies (P = 0.00001). Although LAC were significantly associated with thrombosis in our SLE patients, anti-lamin B1 was not. The frequency of thrombosis in SLE patients expressing both LAC and anti-lamin B1 was similar to that in patients without LAC (P = 1.0). However, patients expressing LAC without anti-lamin B1 had a greater frequency of thrombosis (P = 0.018). CONCLUSION: High titers of IgG anti-lamin B1 autoantibodies are highly specific for a subset of SLE patients whose clinical characteristics include the presence of LAC and other laboratory manifestations of the antiphospholipid syndrome. The presence of LAC without anti-lamin B1 may define a subset of SLE patients at greater risk for thrombosis.