Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo

Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane- proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.     

Tumor necrosis factor-alpha (TNF-alpha) potently enhances in vitro macrophage production from primitive murine hematopoietic progenitor cells in combination with stem cell factor and interleukin-7: novel stimulatory role of p55 TNF receptors

Tumor necrosis factor-alpha (TNF-alpha) is a bifunctional regulator of hematopoiesis, and its cellular responses are mediated by two distinct cell surface receptors. TNF-alpha generally inhibits the growth of primitive murine hematopoietic progenitor cells (Lin-Scal+) in response to multiple cytokine combinations, and the p75 TNF receptor is essential in signaling such inhibition. In the present study we show the reverse phenomenon in that TNF-alpha on the same progenitor cell population in combination with stem cell factor (SCF) and interleukin-7 (IL-7) through the p55 TNF receptor can recruit additional progenitors to proliferate. In contrast, TGF-beta 1, another bifunctional regulator of hematopoietic progenitor cell growth, completely blocked SCF plus IL- 7-induced proliferation. TNF-alpha increased the number of responding progenitors, as well as the size of the colonies formed. The synergistic effects of TNF-alpha were seen at the single cell level, suggesting that its effects are directly mediated. Finally, whereas SCF plus IL-7 promoted primarily granulopoiesis, the addition of TNF-alpha switched the differentiation toward the production of almost exclusively macrophages.     

Demonstration of initial lymphatics in excised human skin using an extension technique and dye injection

Using a newly developed technique (extension technique) we succeeded in presenting dermal lymph vessel networks by subepidermal injection of an aqueous Berlin blue solution in excised human skin. Of 94 skin specimens taken from the safety margin of excised melanomas it was possible to demonstrate 32 networks and 14 single dermal lymph vessels. The networks can be documented by macroscopic photographs. Acetic fixation enables us to produce transparent preparations suitable for investigations with a stereo-microscope. The technique described here for macroscopically demonstrating dermal lymph vessels makes it possible to investigate systemically structural variations of lymph vessel networks in human skin.     

Osteo-radio-necrosis (ORN) and bisphosphonate-related osteonecrosis of the jaws (BRONJ): the histopathological differences under the clinical similarities

Objectives: Both Osteoradionecrosis (ORN) and Bisphosphonate associated osteonecrosis of the jaws (BRONJ) present clinically as regions of exposed necrotic bone. The study aimed to demonstrate the histopathological differences behind the observed clinical similarities. Study Design: Ten ORN specimens and ten BRONJ specimens were used, as well as ten samples of normal mandibular bone as control. Two bone-specific stainings were used, i.e. Sirius Red for the study of the relative presence of collagen types I and III and toluidine blue for the study the osteon density. Results: The Red Green Blue (RGB)-analysis of the specimens stained with Sirius Red identified significant differences between the chromatic patterns observed in bone preparations of patients suffering from ORN when compared to both BRONJ and control samples. Moreover, the osteon density of the BRONJ samples was significantly lower when compared to ORN and normal bone samples. Conclusions: The demonstrated differences in the bone architecture and in the bone collagen content between the two pathological conditions most likely reflect underlying pathophysiological differences.     

Core amino acid variation at position 110 is associated with sustained virological response in Caucasian patients with chronic hepatitis C virus 1b infection

The aim of this study was to analyze the impact of core variations on sustained virological response (SVR) to pegylated interferon plus ribavirin (PEG-IFN/RBV) and its association with predictive factors of response in Caucasian patients infected with genotype 1 hepatitis C virus (HCV-1). Full-length core sequences were analyzed in 100 Caucasian HCV-1-infected patients who received therapy with PEG-IFN/RBV. The associations between variations in the core protein and SVR, as well as with predictors of SVR, were analyzed. Variations at core 62, 70 and 110 were selected as candidates. There were almost no variations at these positions among patients harboring HCV-1a. However, they were identified in 10 (30.3 %), 21 (63.6 %) and 13 (39.4 %) subjects with HCV-1b, respectively. Among the HCV-1b patients, 39.1 % individuals carrying core R62 and 70 % subjects with core R62G showed SVR (p = 0.141), and 66.7 % of HCV-1b patients harboring core R70 and 38.1 % with core R70Q achieved SVR (p = 0.157), whereas the rate of SVR was 70 % for individuals with core T110 and 15.4 % for those with core T110N (p = 0.004). No statistical interaction between core variations and IL28B genotype was observed. Patients with R70 showed higher median (interquartile range) baseline plasma levels of low-density-lipoprotein cholesterol (LDL-C) than those with R70Q (96 [86-118] mg/dL vs. 76 [54-88] mg/dL, p = 0.014). We concluded that a substitution at core 110 is associated with a lower rate of SVR in Caucasian HCV-1b-infected patients receiving PEG-IFN/RBV. Furthermore, the variation at the core 70 position is related to plasma levels of LDL-C in these patients.