Effects of factor VIII concentrates on the immune system of patients with hemophilia

The introduction of high-purity factor VIII (FVIII) concentrates in the treatment of patients with hemophilia A has raised the issue that the use of these products may change on the immune system of the recipients. There is now clear evidence that high-purity concentrates, particularly those produced by immune-affinity chromatography or recombinant DNA technology, slow the fall in CD4 cells that occurs in HIV seropositive patients. It remains to be demonstrated that this biological effect results in clinical benefits and that the occurrence of AIDS is slowed or delayed by the use of high-purity concentrates. On the other hand, concern has been expressed about the possibility that high-purity products might render patients with hemophilia less immunotolerant, facilitating the onset of FVIII antibodies. Follow-up studies of previously untreated hemophiliacs infused for the first time with recombinant FVIII products have ignited this concern, because approximately one fourth of severe hemophiliacs developed inhibitors. However, most of the inhibitors were transient, so that ultimately they had little influence on the efficacy of replacement therapy. It was subsequently realized that inhibitors develop with high frequency even in hemophiliacs treated with less pure, plasma-derived products, provided testing is prospective and as frequent as for studies of recombinant FVIII. On the whole, these data have provided new insights on the natural history of inhibitor development in previously untreated hemophiliacs, showing that low-titer, short-lasting inhibitors develop more frequently than previously recognized.     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium

To establish the process by which transplanted cells integrate into the liver parenchyma, we used dipeptidyl peptidase IV-deficient F344 rats as hosts. On intrasplenic injection, transplanted hepatocytes immediately entered liver sinusoids, along with attenuation of portal vein radicles on angiography. However, a large fraction of transplanted cells (>70%) was rapidly cleared from portal spaces by phagocyte/macrophage responses. On the other hand, transplanted hepatocytes entering the hepatic sinusoids showed superior survival. These cells translocated from sinusoids into liver plates between 16 and 20 hours after transplantation, during which electron microscopy showed disruption of the sinusoidal endothelium. Interestingly, production of vascular endothelial growth factor was observed in hepatocytes before endothelial disruptions. Portal hypertension and angiographic changes resulting from cell transplantation resolved promptly. Integration of transplanted hepatocytes in the liver parenchyma required cell membrane regenesis, with hybrid gap junctions and bile canaliculi forming over 3 to 7 days after cell transplantation. We propose that strategies to deposit cells into distal hepatic sinusoids, to disrupt sinusoidal endothelium for facilitating cell entry into liver plates, and to accelerate cell integrations into liver parenchyma will advance applications of hepatocyte transplantation.     

Contribution of NMDA and non-NMDA receptors to synaptic transmission from the brachium of the inferior colliculus to the medial subdivision of the medial geniculate nucleus in the rabbit

Previous work from this laboratory has demonstrated that monosynaptic inputs from the brachium of the inferior colliculus (BIC) to the medial subdivision of the medial geniculate nucleus (mMG) strengthen as a result of associative conditioning with an acoustic conditioned stimulus (i.e., fear conditioning). One model that has been proposed to underlie certain types of neuronal plasticity involves the recruitment of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors. The purpose of the present study was to examine the relative contributions of glutamatergic NMDA and non-NMDA receptors to synaptic transmission within this pathway. Individual contributions of the specific receptor types were assessed through the use of 2-amino-5-phosphonovaleric acid (AP5), a selective NMDA receptor antagonist, and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. Bipolar stimulating electrodes were stereotaxically implanted in BIC and recording electrodes (attached to dual 32-gauge cannulae for delivery of drug) were positioned in mMG of New Zealand albino rabbits. Single pulses (150 micros, 100-350 microA) delivered to BIC resulted in short-latency (<4 ms) responses in mMG. BIC-evoked single-unit activity was recorded from mMG before, during, and at several intervals after injection of AP5, CNQX, and/or artificial cerebrospinal fluid (ACSF). Injection of either AP5 or CNQX, but not ACSF, significantly attenuated the short-latency BIC-evoked responses in the vast majority of cells tested. These findings suggest that the monosynaptic pathway from BIC to mMG is glutamatergic and that this pathway frequently employs NMDA-type receptors during electrically stimulated synaptic transmission. Due to the NMDA receptors' proposed role in plasticity (e.g., long-term potentiation), these results may have implications for understanding the mechanisms of synaptic plasticity observed at this synapse during associative learning.     

Ionic mechanisms of action of neurotensin in acutely dissociated neurons from the diagonal band of Broca of the rat

Whole cell recordings were performed on acutely dissociated neurons from the horizontal limb of the diagonal band of Broca (hDBB) from rats to elucidate the ionic mechanisms of action of neurotensin. Neurotensin caused a decrease in whole cell voltage-activated outward currents and failed to elicit a response when Ca2+ influx was blocked by changing the external solution to the one containing 0 mM Ca2+ and 50 microM Cd2+, suggesting the involvement of Ca2+-dependent conductances. Charybdotoxin, a specific blocker of voltage-sensitive calcium-activated K+ channels (IC), caused a decrease in outward currents comparable with that caused by blocking calcium influx and occluded the neurotensin-induced decrease in outward currents. Similarly, 50 microM tetraethylammonium ions also blocked the neurotensin response. Also neurotensin reduced whole cell barium currents (IBa) and calcium currents (ICa). Amiloride and omega-conotoxin GVIA, but not nimodipine, were able to eliminate the neurotensin-induced decrease in IBa. Thus T- and N- but not L-type calcium channels are subject to modulation by neurotensin, and this may account for its effects on IC. The predicted changes in action potential as a result of the blockade of currents through calcium channels culminating into changes in IC were confirmed in the bridge current-clamp recordings. Specifically, neurotensin application led to depolarization of the resting membrane potential, broadening of spike and a decrease in afterhyperpolarization and accommodation. These alterations in action potential characteristics that resulted in increased firing rate and excitability of the hDBB neurons also were produced by application of charybdotoxin. Neurotensin effects on these properties were occluded by 2 - [(1 - 7 - chloro - 4 - quinolinyl) - 5 - (2, 6 - di - methoxyphenyl) pyrazol-3-yl) carbonylamino] tricyclo (3.3.1.1.)decan-2-carboxylic acid, a nonpeptide high-affinity neurotensin receptor antagonist. Neurotensin blockade of IC, possibly through ICa, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm.