CD71 antibody enhances iron uptake by mouse bone marrow cells and the survival potential of erythroid progenitor cells.

Transferrin has been shown to have cytoprotective functions. The aim of this study was to evaluate the effect of antibody against CD71 (transferrin receptor) on survival potential of erythroid progenitor cells. After anti-CD71 IgG or control IgG was reacted with cells for 20 min on ice, the uptake of transferrin-bound 59Fe by the MEL cells or the mouse bone marrow cells during 2-day incubation without erythropoietin was measured. Erythropoiesis was evaluated by addition of erythropoietin after 4-5 weeks' culture without erythropoietin or beta-mercaptoethanol, and production of cells containing hemoglobin was compared by immunocytological methods. MEL cells reacted with anti-CD71 IgG incorporated more transferrin-bound 59Fe than MEL cells reacted with control IgG. Uptake of transferrin-bound 59Fe by mouse bone marrow cells reacted with anti-CD71 IgG was also greater than by cells reacted with control IgG. Erythrocytes developed from cells reacted with anti-CD71 IgG and cultured for 4-5 weeks, but few erythrocytes developed from cells reacted with control IgG. Bone marrow cells depleted of TER119+ cells, CD34+ cells, or CD45+ cells and reacted with CD71 antibody did not differentiate into erythrocytes. Survival potential of erythroid progenitor cells were enhanced by reaction with anti-CD71 antibody.

Pyrogenic cytokines injected into the rat cerebral ventricle induce cyclooxygenase-2 in brain endothelial cells and also upregulate their receptors.

Peripheral immunological insults induce interleukin (IL)-1 beta and IL-6 in the brain. To elucidate the mechanism(s) of fever evoked by these brain-derived cytokines, and possible interactions between them, we examined in rats: (i) whether cyclooxygenase-2 is responsible for fever evoked by central injection of these cytokines; (ii) if so, where in the brain cyclooxygenase-2 is induced; (iii) where the receptors for these cytokines are located; and (iv) how the expression of these receptors is influenced by the cytokines. Intracerebroventricular injection of these cytokines evoked fever that was suppressed by a cyclooxygenase-2 inhibitor. Brain endothelium was the site of cyclooxygenase-2 induction by these cytokines. IL-1 receptor (IL-1R) was constitutively expressed in brain endothelium, and its mRNA was further upregulated by either cytokine. IL-6R mRNA was constitutively expressed in the cerebral cortex, and was newly induced in as yet unidentified cells in brain blood vessels by either cytokine. Messenger RNAs for cyclooxygenase-2, IL-1R, and IL-6R were often observed in the same blood vessels. These results suggest that COX-2 induced in brain endothelium is, at least in part, involved in the fever evoked by these cytokines, and that one possible interaction between these two cytokines is mutual upregulation of their receptors in the endothelium or perivascular cells, resulting in augmentation of their actions.

Immunohistochemical investigation of caspase-1 and effect of caspase-1 inhibitor in delayed neuronal death after transient cerebral ischemia.

The localization of caspase-1 protein, interleukin-1beta (IL-1beta)-converting enzyme, was immunohistochemically examined in the hippocampal CA-1 subfield by a transient occlusion of bilateral common carotid arteries in Mongolian gerbils. Immunoreactivities for caspase-1 were found in microglias, astrocytes, endothelial cells of capillaries and some non-pyramidal neurons. Immunopositive microglias increased in number from 3 days until 7 days from the transient ischemia, and astrocytes also increased in number from 3 days until 28 days. At the electron microscopic level, caspase-1 immunoreaction endproducts were associated with Golgi apparatus in glial cells, endothelial cells of blood vessels and non-pyramidal neurons. The delayed neuronal death of CA-1 pyramidal cells was significantly protected by the treatment of specific caspase-1 inhibitor (Ac-WEHD-CHO) or broad caspase family inhibitor (z-VAD-FMK). Cell death was protected in a dose dependent manner by the former by 43-57%, and by the latter by 66-91% when injected at 1 and 10 microg, respectively. On the other hand, the protective effect of specific caspase-3 inhibitor (Ac-DMQD-CHO) was less significant at higher dose (10 microg) by 33% (P<0.05), and not detectable at lower dose (1 microg) by 13% (P=0.27). Furthermore, a significant decrease of microglias and astrocytes was found in the CA-1 as well as the reduction of IL-1beta and caspase-1 immunoreactivities by the treatment of Ac-WEHD-CHO. Extravasation of serum albumin was also extremely reduced by this treatment. These findings suggest that the inhibition of caspase-1 activity ameliorates the ischemic injury by inhibiting the activity of IL-1beta.

Rescue of ischemic brain injury by adenoviral gene transfer of glial cell line-derived neurotrophic factor after transient global ischemia in gerbils.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor (TGF)-beta superfamily, is one of the most potent neurotrophic factors and promotes survival of many populations of cells. We examined neuroprotective effect of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the transient global ischemia. Gerbils received administration of AxCAhGDNF or an adenoviral vector encoding bacterial beta-galactosidase gene (AxCALacZ) through the lateral ventricle. Two days later, occluding bilateral common carotid arteries for 5 min using aneurysm clips produced the transient global forebrain ischemia. Animals showed intense immunolabeling for GDNF in ependymal cells on 2, 4 and 7 days after the operation. The exogenous gene transducted by adenovirus in the same cells was detected by in situ hybridization. The treatment with AxCAhGDNF significantly prevented the loss of hippocampal CA1 pyramidal neurons 2 to 7 days after the operation, as compared to AxCALacZ treatment. Also terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) staining was markedly reduced in the case with AxCAhGDNF treatment at 7 days after the operation. These results indicated that the adenovirus-mediated gene transfer of GDNF might prevent the delayed neuronal death of stroke and other disorders of the cerebral vasculature.

TGF-beta1 produced by gastric cancer cells affects mesothelial cell morphology in peritoneal dissemination.

In vitro morphologic change of mesothelial cells was observed following the addition of serum-free conditioned medium (SF-CM) from peritoneal dissemination cell line OCUM-2MD3. The same morphologic change of mesothelial cells was observed following the addition of 10 ng/ml TGF-beta1, but not following the addition of b-FGF, IGF-I, VEGF or PDGF-AA. In the in vivo study, mesothelial cells of mice treated with SF-CM from OCUM-2MD3 and TGF- beta1 were separated from one another, resulting in exposure of the submesothelial connective tissue. The molecular size of the mesothelial morphology changing activity was estimated by running the SF-CM from OCUM-2MD3 through a gel filtration column TSK-gel G2000SW. The mesothelial morphology changing activity was recognized at positions equivalent of Mr 6, 500-30,000. 25 kDa TGF-beta1 was detected in the active fraction from the TSK-gel G2000SW column and the SF-CM of OCUM-2MD3 by Western blotting using a monoclonal antibody against TGF-beta1. These findings suggest that TGF-beta1 produced by gastric cancer cells changes the morphology of mesothelial cells and may thus be closely associated with peritoneal dissemination.

Single stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in the gerbil hippocampus.

The light and electron microscopic localizations of single stranded DNA (SSD) protein, a marker of apoptosis and programmed cell death, in the gerbil hippocampus were examined by immunocytochemistry after transient brain ischemia. SSD-immunoreactive (IR) cells appeared from post-operative day 1 (PO 1) to PO 7 after 5- or 10-min ischemia. Immunoreaction was recognized in the nucleus of the CA1 pyramidal neurons without remarkable morphological changes on PO 1. These findings suggest that SSD degradation can occur during delayed neuronal death in the CA1, preceding the appearance of double strand breaks, one of the characteristic features of apoptosis.