ß2-adrenergic receptors protect axons during energetic stress but do not influence basal glio-axonal lactate shuttling in mouse white matter.

In vitro studies have demonstrated that ß2-adrenergic receptor activation stimulates glycogen degradation in astrocytes, generating lactate as a potential energy source for neurons. Using in vivo microdialysis in mouse cerebellar white matter we demonstrate continuous axonal lactate uptake and glial-axonal metabolic coupling of glutamate/lactate exchange. However, this physiological lactate production was not influenced by activation (clenbuterol) or blocking (ICI 118551) of ß2-adrenergic receptors. In two-photon imaging experiments on ex vivo mouse corpus callosum subjected to aglycemia, ß2-adrenergic activation rescued axons, whereas inhibition of axonal lactate uptake by α-cyano-4-hydroxycinnamic acid (4-CIN) was associated with severe axonal loss. Our results suggest that axonal protective effects of glial ß2-adrenergic receptor activation are not mediated by enhanced lactate production.

Neuroprotective efficacy of subcutaneous insulin-like growth factor-I administration in normotensive and hypertensive rats with an ischemic stroke.

The aim of this study was to test the insulin-like growth factor-I (IGF-I) as a neuroprotective agent in a rat model for ischemic stroke and to compare its neuroprotective effects in conscious normotensive and spontaneously hypertensive rats. The effects of subcutaneous IGF-I injection were investigated in both rat strains using the endothelin-1 rat model for ischemic stroke. Motor-sensory functions were measured using the Neurological Deficit Score. Infarct size was assessed by Cresyl Violet staining. Subcutaneous administration of IGF-I resulted in significantly reduced infarct volumes and an increase in motor-sensory functions in normotensive rats. In these rats, IGF-I did not modulate blood flow in the striatum and had no effect on the activation of astrocytes as assessed by GFAP staining. In hypertensive rats, the protective effects of IGF-I were smaller and not always significant. Furthermore, IGF-I significantly reduced microglial activation in the cortex of hypertensive rats, but not in normotensive rats. More detailed studies are required to find out whether the reduction by IGF-I of microglial activation contributes to an impairment IGF-I treatment efficacy. Indeed, we have shown before that microglia in hypertensive rats have different properties compared to those in control rats, as they exhibit a reduced responsiveness to ischemic stroke and lipopolysaccharide.

Tec kinase stimulates cell survival in transfected Hek293T cells and is regulated by the anti-apoptotic growth factor IGF-I in human neutrophils.

OBJECTIVE: Previously, we showed that the phosphatidylinositol-3 kinase (PI(3)K) pathway mediates the anti-apoptotic effects of IGF-I in human neutrophils independently of its down-stream target Akt. In this study, we investigated whether IGF-I regulates Tec kinase, an alternative down-stream target of PI(3)K, in neutrophils and whether this molecule is able to affect apoptosis. DESIGN: We investigated the translocation of Tec kinases in neutrophils after stimulation with IGF-I. Furthermore, we transiently and stably transfected Hek293T cells with constructs expressing different forms of Tec kinase and measured the level of cell survival and apoptosis/necrosis through trypan blue exclusion test and Annexin-V/propidium iodide labelling, respectively. RESULTS: We show that IGF-I stimulates the translocation of Tec kinase to the membrane in neutrophils in a PI(3)K dependent matter. Overexpression of Tec kinase augments cell survival by inhibition of necrosis. The pro-survival effect is attenuated by the deletion of the kinase domain but not by inactivation of this domain by a single amino acid substitution. CONCLUSION: Tec kinase can act as a prosurvival factor and is regulated by IGF-I in human neutrophils through PI(3)K activation.

An extraordinary cause for deep venous thrombosis.

The authors present a case of a congenital absence of the infrarenal inferior vena cava in an 18-year-old man showing symptoms of deep venous thrombosis of the left leg. The congenital absence of the inferior vena cava is typically asymptomatic and is commonly reported as a fortuitous finding. Abnormalities of the inferior vena cava are risk factors contributing to the development of deep venous thrombosis. The absence of vena cava is underestimated in patients with deep venous thrombosis because in some cases compression B-mode ultrasonography will not reveal the condition. CT should be made available for all young patients with idiopathic deep venous thrombosis.

"Twist of the wrist" a rare case of carpal dislocation.

A rare case of a scaphoid-trapezium dislocation is presented. The treatment was open reduction, ligament repair, and internal fixation with a Kirschner wire. After 4 weeks of immobilization, the Kirschner wire was removed, and full recovery was obtained 12 weeks after the trauma.

Multiple cAMP-induced signaling cascades regulate prolactin expression in T cells.

Beside its pivotal role in reproduction, the pituitary hormone prolactin (PRL) has been attributed an immunomodulatory function. Here we report that cAMP is an important stimulator of PRL transcription in primary human T lymphocytes. Inhibition of both protein kinase A (PKA) and p38 MAPK partially abrogated cAMP-induced PRL expression. In addition, cAMP-induced phosphorylation of p38 was shown to occur independently of PKA and could be mimicked by a methylated cAMP analogue which specifically activates the recently discovered cAMP receptor EPAC (exchange protein directly activated by cAMP). Our findings suggest that cAMP induces PRL expression in T lymphocytes via cooperation of at least two different signaling pathways: a PKA-dependent pathway leading to the phosphorylation of cAMP response element-binding protein, and a PKA-independent pathway leading to p38 phosphorylation.

Specific roles for the PI3K and the MEK-ERK pathway in IGF-1-stimulated chemotaxis, VEGF secretion and proliferation of multiple myeloma cells: study in the 5T33MM model.

Insulin-like growth factor-1 (IGF-1) has been described as an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Angiogenesis correlates with development and prognosis of the MM disease. Vascular endothelial growth factor (VEGF) is one of the prominent factors involved in this process. The different functions of IGF-1 were investigated in the 5TMM mouse model with emphasis on proliferation, migration and VEGF secretion, and the signalling pathways involved. Western Blot analysis revealed that ERK1/2 and Akt (PKB) were activated after IGF-1 stimulation. The activation of ERK1/2 was reduced by the PI3K inhibitor Wortmannin, implying that the PI3K pathway is involved in its activation. Insulin-like growth factor-1 induced an increase in DNA synthesis in MM cells, which was mediated by a PI3K/Akt-MEK/ERK pathway. Insulin-like growth factor-1 enhanced F-actin assembly and this process was only PI3K mediated. Stimulation by IGF-1 of VEGF production was reduced by PD98059, indicating that only the MEK-ERK pathway is involved in IGF-1-stimulated VEGF production. In conclusion, IGF-1 mediates its multiple effects on MM cells through different signal transduction pathways. In the future, we can study the potential in vivo effects of IGF-1 inhibition on tumour growth and angiogenesis in MM.

Myeloid leukemic cells express and secrete bioactive pituitary-sized 23 kDa prolactin.

Prolactin (PRL) is a 23 kDa polypeptide hormone of pituitary origin which is of major importance for reproduction. In addition, PRL has immunomodulatory effects and can be produced in small quantities in nonpituitary tissues. To address possible autocrine or paracrine functions of PRL in leukemia, we characterized immunoreactive PRL from the culture medium of leukemic cells. The myeloid cell line Eol-1 expresses the long extrapituitary type mRNA for PRL and synthesizes immunoreactive PRL with a molecular weight of 23 kDa. The biological activity in Eol-1 culture medium was determined using the Nb2 bioassay. This activity co-eluted with recombinant human (rh) PRL on an S-200 Sephacryl gel filtration column and could be blocked by anti-PRL antiserum. Western blot analysis and Nb2 bioassays also suggest that acute myelogenous leukemic blasts secrete bioactive 23 kDa PRL in one out of three tested patients.

Growth hormone and prolactin expression in the immune system.

Prolactin (PRL) and growth hormone (GH) are pituitary hormones that play pivotal roles in lactation and body growth, respectively. In addition, both hormones have been implicated as modulators of immune responses. Since the expression of GH and PRL by leukocytes points to autocrine or paracrine roles during immune responses, our study is aimed at PRL- and GH-production in leukocytes. We show that human peripheral blood granulocytes, which express GH and PRL mRNA, contain high molecular-weight immunoreactive variants of GH and PRL (37 and 43 kDa, respectively), but not the pituitary-sized hormones. Secretion of these variants, or biologically active material as assessed by the Nb2 bioassay, was not detected. On the other hand, certain leukemic myeloid cells secrete 23-kDa, pituitary-sized, PRL, which is biologically active.

The role of growth hormone and insulin-like growth factors in the immune system.

Growth hormone (GH) and insulin-like growth factor I (IGF-I) can modulate the development and function of the immune system. In this chapter, we present data on the expression of receptors for GH and IGFs and the in vitro and in vivo effects of these proteins. We show that expression of GH and IGFs in the immune system opens up the possibility that these proteins are not only involved in endocrine control of the immune system but can also play a role as local growth and differentiation factors (cytokines). Endocrine control of GH could be direct or mediated via endocrine or autocrine/paracrine IGF-I. In addition, GH can act as an autocrine or paracrine factor itself. Furthermore, IGF-I in the immune system has been shown to be regulated by cytokines, such as interleukin-1 and interferon-gamma, alluding to a cytokine-like function of IGF-I. In addition to data on the function of GH and IGF-I in the immune system, we present new findings which imply a possible function of IGF-II and IGF-binding proteins.

T and B cell development in pituitary deficient insulin-like growth factor-II transgenic dwarf mice.

Treatment of mice with IGF-I stimulates T and B cell development. We showed that overexpression of IGF-II in transgenic FVB/N mice only stimulated T cell development. In the present study, we further addressed the in vivo effects of IGF-II in the absence of IGF-I to get more insight into the potential abilities of IGF-II to influence T and B cell development. To this end, we studied lymphocyte development in IGF-II transgenic Snell dwarf mice that are prolactin, GH and thyroid-stimulating hormone deficient and as a consequence show low serum IGF-I levels. We showed that T cell development was stimulated to the same extent as in IGF-II transgenic FVB/N mice. Furthermore, IGF-II increased the number of nucleated bone marrow cells and the number of immature B cells without having an effect on the number of mature B cells in spleen and bone marrow. Our data show that IGF-II has preferential effects on T cell development compared with B development, and that these preferential effects also occur in the absence of measurable IGF-I levels.

Human neutrophils express GH-N gene transcripts and the pituitary transcription factor Pit-1b.

Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 +/- 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but not the GH-V gene. Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b.

Growth hormone expression in murine bone marrow cells is independent of the pituitary transcription factor Pit-1.

GH has been shown to promote the development and function of leukocytes. The expression of both GH and GH-receptors in lymphoid cells has led to the hypothesis that GH acts in an autocrine or paracrine fashion. The described effects of GH on hematopoiesis and B cell development, led us to investigate GH expression in bone marrow cells. By immunocytochemistry, we show that bone marrow-derived granulocytes and macrophages contain immunoreactive GH. We found that 65 +/- 24% of the granulocytes were stained with anti-GH, whereas 5.8 +/- 1.5% of the granulocytes contained detectable amounts of GH mRNA as assessed by in situ hybridization. To address a possible alternative regulation mechanism in bone marrow and to establish whether locally derived GH might still play a role in pituitary-deficient dwarf mice, we also addressed GH expression in bone marrow from hypopituitary Snell dwarf mice. These mice have a mutated gene for the pituitary transcription factor Pit-1 that is deficient in DNA binding. Our finding that GH expression (immunoreactive protein and mRNA) in bone marrow cells from dwarf mice is similar to that in normal mice points to a Pit-1 independent regulation of GH in mouse bone marrow.

T cell subsets and T cell function in cartilage-hair hypoplasia.

Cartilage hair hypoplasia is a rare autosomal recessive form of short-limbed dwarfism associated with a cellular immunodeficiency. In eight patients, the authors studied the presence of T cell subsets and in vitro T cell function in order to address the basis for the immunological disorder. Both the proliferative response to phytohaemagglutinin (PHA) and the PHA-induced IL2 production were 60% lower compared with controls (P = 0.007 and 0.005, respectively). The impaired proliferative response could not be restored by addition of IL-2. This result is in accordance with a decrease in the percentage of activated T cells expressing the p55 subunit of the IL-2 receptor complex (CD25). The results define more precisely that T cells from cartilage hair hypoplasia patients are defective in the transition from the G0 to the G1 phase of the cell cycle. Furthermore, the data demonstrate that several CHH patients show a reduced proportion of CD45RA+ 'naive' T cells. However, the in vitro impairment of T cell function cannot solely be explained by imbalance between 'naive' and 'memory' T cells. Although CHH patients with a history of recurrent respiratory tract infections showed the most aberrant in vitro immune parameters, a clear relationship between clinical data and in vitro parameters could not be established for the whole patient group.

IGF-I potentiates interleukin-2 production in human peripheral T cells.

IGF-I stimulates the proliferation and differentiation of many cell types. In the case of T cells, IGF-I has been described to potentiate mitogen-induced DNA synthesis. We have addressed the working mechanism of IGF-I on T cell proliferation by measuring the effects of IGF-I on various stages of T cell activation. We found that IGF-I augmented the phytohaemagglutinin- and anti-CD3-induced interleukin-2 (IL2) production of human peripheral T cells before they enter the S phase of the cell cycle. Furthermore, the addition of IGF-I did not influence DNA synthesis of IL2-dependent growing T cells.

Type I insulin-like growth factor receptor expression in different developmental stages of human thymocytes.

Insulin-like growth factor-I (IGF-I) has been implicated in playing a regulatory role in T cell development and in T cell function. We demonstrate the presence of type I IGF receptors on human thymocytes using radioligand binding assays and flowcytometric analysis. The relative potencies of IGF-I, IGF-II and insulin for competition with 125I- IGF-I indicate the presence of type I IGF receptors. Scatchard analysis revealed that the average number of receptors per thymocyte is 257 +/- 28 with a Kd of 0.12 +/- 0.01. With multicolour flowcytometry using a type I IGF receptor specific monoclonal antibody (alpha IR3), we show that CD4-CD8- cells express 3-4 times more receptors per cell as compared with CD4+CD8+, CD4+CD8- and CD4-CD8+ cells. IGF-I directly stimulated DNA synthesis of thymocytes and potentiated DNA synthesis in mitogen-activated thymocytes. These results indicate that IGF-I can influence T cell development in humans at the level of the thymus.

Insulin-like growth factor induces phosphorylation of immunoreactive insulin receptor substrate and its association with phosphatidylinositol-3 kinase in human thymocytes.

Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1-transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160-180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF-I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells.