Eva-Maria Wolber

The kidney as a second site of human C-reactive protein formation in vivo.

C‐reactive protein (CRP) is the main acute phase reactant in humans. Its production is presumably restricted to the liver but extrahepatic expression by inflamed tissue has not been studied indetail. By real‐time PCR and immunohistochemistry we here show that renal cortical tubular epithelial cells (TEC) express CRP mRNA and protein within 6 h after stimulation with conditioned medium (CM) or IL‐6, but not IL‐1α or TNF‐α. Western blot analysis with monoclonal anti‐CRP antibody that recognizes native CRP revealed protein secretion into supernatants of CM‐stimulated TEC cultures. While hepatoma‐derived Hep3B cells could be induced similarly, peripheral blood mononuclear cells could not. CRP mRNA transcripts were observed in nephrectomized renal allografts with severe acute rejection but not with chronic allograft nephropathy (CAN). Of 19 needle biopsies of acutely rejecting kidney transplants, 15 demonstrated CRP mRNA production with the relative expression levels increasing with the severity of rejection. On the other hand, none of 7 graft biopsies with acute tubular necrosis (ATN) or CAN showed CRP mRNA expression. By using monoclonal anti‐CRP antibody, cortical tubules as well as glomerular cells were shown to locally express CRP in rejecting, but not in ATN kidneys. We conclude that inflamed kidneys represent a so far unknown site of CRP formation in vivo. These data shed new light on the acute phase reaction not merely representing a systemic inflammatory pathway but probably being part of the local immune response.

Erythropoietin gene expression in different areas of the developing human central nervous system

UNLABELLED Evidence from cell culture and animal experiments suggests a neuroprotective and neurotrophic function of erythropoietin (EPO). We have quantitated the distribution of EPO mRNA expression in the developing human central nervous system (CNS). PATIENTS AND METHODS Up to seven biopsies from different areas of the CNS of four preterm fetuses (gestational age 23-37 weeks) were obtained at routine postmortem examinations. EPO mRNA was quantitated by competitive PCR in samples from the CNS, the kidneys, and the liver where the EPO gene is predominantly expressed at this gestational age. RESULTS EPO mRNA was most abundant in one sample from the cerebellum (0.29 amol/microg total RNA [amol=10(-18)mol]) and two from the pituitary gland (0.23 amol/microg total RNA), but levels varied considerably. EPO mRNA in the cortex cerebri (median 0.12 amol/microg total RNA; n=4) dominated over the expression in the corpora amygdala (median 0.05 amol/microg total RNA; n=4), the hippocampus (median 0.03 amol/microg total RNA; n=4), or the basal ganglia (median 0.01 amol/microg total RNA; n=3). Only little EPO mRNA (<0.01 and 0.06 amol/microg total RNA) was found in the spinal cord. EPO mRNA levels in the cerebellum, pituitary gland, or the cerebral cortex were within the same range as in the liver (0.03-1.67 amol/microg total RNA; n=4), or the kidneys (0.06-0.79 amol/microg total RNA; n=4). CONCLUSION We found the EPO gene expressed throughout the fetal human CNS. Our data provide the basis to discuss a function for EPO in the brain of humans as well.

Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B.

The concentration of circulating thrombopoietin (TPO) is relatively high in patients with thrombocytosis reactive to inflammatory diseases. We investigated whether immunomodulatory cytokines stimulate TPO synthesis in cultured human hepatoma cells (lines HepG2 and Hep3B), renal proximal tubular cells, and bone marrow fibroblasts. The effects of interleukins (IL) IL-1beta, IL-6, and IL-11 and of tumor necrosis factor-a (TNF-alpha) on the rate of TPO secretion were measured by ELISA. TPO mRNA levels were quantitated by competitive reverse transcription PCR. HepG2 and Hep3B cells produced significant amounts of TPO mRNA and TPO protein. Renal tubular cells synthesized less TPO, and in bone marrow fibroblasts, neither TPO mRNA nor TPO protein was detected. Only IL-6 affected TPO protein secretion, causing a 1.5-fold stimulation in HepG2 and Hep3B cells in 24-h incubation periods. The TPO mRNA content in these cells was doubled by IL-6 after 2, 6, or 24 h of stimulation. Thus, IL-6 could cause thrombocytosis in inflammatory disease partly by increasing hepatic TPO production.

Thrombopoietin gene expression in the developing human central nervous system.

Thrombopoietin gene expression in the human adult central nervous system (CNS) appears to be locally restricted. The aim of this study was to identify areas of thrombopoietin expression in the developing human CNS, and to compare the thrombopoietin mRNA content in the CNS to that in liver and kidneys as major sites of thrombopoietin production. Thrombopoietin protein concentrations in the cerebrospinal fluid (CSF) were measured by ELISA. In 14 fetuses and neonates with perinatal death, thrombopoietin mRNA expression was measured by competitive RT-PCR. Thrombopoietin mRNA was expressed in 29 of 32 specimens taken from the CNS. The following ranking of the intensity of expression in the CNS was possible: Spinal cord=cerebellum=cortex>>pituitary gland>>>brain stem=corpora amygdala=hippocampus. Whereas in the latter three tissues only trace amounts of thrombopoietin transcripts were detectable, thrombopoietin mRNA levels in the spinal cord were comparable to levels in liver and kidney. Thrombopoietin protein concentrations in CSF ranged between 41 and 75 pg/ml. In the developing human CNS, the thrombopoietin gene is abundantly expressed. Considering that thrombopoietin contains a neurotrophic sequence, it may well play a role in neuronal cell biology.

Vascular endothelial growth factor mRNA in eutopic and ectopic endometrium

OBJECTIVE To evaluate changes in expression levels of vascular endothelial growth factor (VEGF) mRNA in human endometrial explants in a chicken chorioallantoic membrane model of endometriosis. DESIGN Experimental prospective study. SETTING University hospital. PATIENT(S) Endometrial biopsy samples were obtained from healthy, ovulating women undergoing elective surgery. INTERVENTION(S) Endometrial fragments were placed on the chicken chorioallantoic membrane and removed for analysis after 0, 24, 48, and 72 hours. MAIN OUTCOME MEASURE(S) Expression of different VEGF mRNA splice variants was tested. Expression of VEGF(165) mRNA was assessed by using competitive polymerase chain reaction and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. RESULT(S) After 0, 24, 48, and 72 hours of incubation, all grafts expressed VEGF(121), VEGF(145), VEGF(165), and VEGF(189) mRNA. Expression of VEGF(165) mRNA increased up to 10-fold at 24 to 72 hours compared with precultivation values. CONCLUSION(S) Levels of VEGF(165) mRNA in endometrial grafts increase after implantation on chicken chorioallantoic membrane. Hypoxic induction of VEGF mRNA expression in endometrial cell cultures has been reported previously. Induction of VEGF expression might indicate relative hypoxia of the specimen due to insufficient vascularization. Expression of VEGF may assist in vascularization of endometrial explants after retrograde menstruation.

Short Communication: Thrombopoietin Production in Human Hepatic Cell Cultures (HepG2) Is Resistant to IFN-α, IFN-β, and IFN-γ Treatment

Thrombocytopenia is an important complication of interferon (IFN) therapy for chronic viral hepatitis. To study whether IFN interferes with hepatic thrombopoietin (TPO) synthesis, we used the human hepatoma cell line HepG2. Our results show that IFN-α, IFN-β, or IFN-γ did not impair TPO mRNA expression, as determined by quantitative RT-PCR, even when high IFN doses (up to 5000 U/ml) or long-term incubations (up to 14 days) were applied. Neither was the rate of secretion of immunoreactive TPO reduced on IFN treatment. These findings support the concept that IFNs primarily mediate effects on megakaryocytic cells and platelets rather than on TPO-producing hepatocytes.