Pharmacokinetics of LJP 993, a tetrameric conjugate of domain 1 of beta2-glycoprotein I for antiphospholipid syndrome.

beta2-glycoprotein I is the best-characterized antigenic target for antiphospholipid autoantibodies. We synthesized a tetrameric conjugate of the domain 1 of beta2-glycoprotein I (LJP 993) aimed at developing the conjugate as a Toleragen to suppress antiphospholipid syndrome. The present studies focused on determining the stability, tissue distribution, plasma concentration-time profile and excretion of the LJP 993 in mice. The stability of LJP 993 in mouse plasma was quantitatively evaluated using strong cation-exchange high performance liquid chromatography. ( 125)I-labeled LJP 993 was intravenously injected to mice, and levels of (125)I-labeled LJP 993 in plasma, tissues, urine and feces were determined at known intervals. Incubation of LJP 993 with mouse serum at 37 degrees C for 8 h resulted in a decrease by 34% of LJP 993 concentration. No degradation fragment was observed during the incubation. After a single intravenous administration of (125)I-LJP 993 (0.5 and 5 mg/kg) to mice, both C(max) and area-under-curve values increased in a dose-proportional manner, and blood radioactivity disappeared in a bi-exponential manner with the distribution half-lives equal to 1.7 min, and the elimination half-lives 188 and 281 min, respectively. The (125)I-LJP 993 was moderately distributed into organs and tissues with the exception that brain level of ( 125)I-LJP 993 was negligible. The major sites of (125)I-LJP 993 uptake were the kidney (at 30 min post dosing), and kidney, lung, liver, heart, spleen, skin, muscle and fat tissues (at 4 h post dosing). Cumulative urinary and fecal radioactivity for 0-48 h post dosing accounted for 44.7% and 4.2% of the administered dose, respectively, with the fast rate of urinal excretion occurring within the first 8 h. In summary, LJP 993 was fairly stable in mouse plasma. After administration to mice, (125)I-LJP 993 was taken up mainly by kidney and then distributed extensively to tissues except brain. Both C(max) and area-under-curve values increased in a dose-proportional manner. It was predominantly excreted in the urine with an elimination half-life longer than 3 h. Kidney is a major route to excrete the tetrameric conjugate.

Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-gamma-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression.

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon-gamma (IFN-gamma)-induced priming of macrophages (m phi s) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal m phi s from Lewis (LEW/N) rats for 18 h with recombinant rat IFN-gamma (rRaIFN-gamma) in the presence of either adenosine (100 microM) or MDL 201,112 (10 microM) resulted in a fourfold decrease in superoxide anion (O2-) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN-gamma treatment. Peritoneal m phi s from LEW/N rats stimulated with LPS/rRaIFN-gamma were observed to secrete immunoreactive and bioactive TNF-alpha over 18 h in vitro and this cytokine could be dose-dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of O2- release mediated by MDL 201,112. Furthermore, incubation of LEW/N m phi s for 18 h in vitro with rRaIFN-gamma resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 microM) or adenosine (100 microM). MDL 201,112 and adenosine were also effective in decreasing m phi opsonized zymosan-stimulated O2- levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down-regulation of heat-killed M. tuberculosis-activated LEW/N m phi MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat m phi activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases.

Effect of hemoglobin on neurogenic responses and cholinergic parameters in porcine cerebral arteries.

Electrically stimulated neurogenic vasodilation and endothelial-dependent cholinergic vasodilation in cerebral arteries are both blocked by hemoglobin. To determine if neurogenic vasodilation has a cholinergic component, we examined the effect of hemoglobin on neurogenic responses and perivascular cholinergic parameters in isolated porcine cerebral arteries. The perfused circle of Willis has a mixed response to transmural nerve stimulation (TNS) that is predominantly vasodilation. Exposure to hemoglobin (5 microM) causes constriction of this preparation while simultaneously blocking TNS-induced vasodilation. At similar concentrations, however, hemoglobin did not alter electrically stimulated, tetrodotoxin-sensitive release of acetylcholine. Hemoglobin also had no effect on neuronal choline uptake or esteratic inactivation of acetylcholine. These results demonstrate the ability of low concentrations of hemoglobin to alter cerebral neurogenic vasodilation. The failure of hemoglobin to affect any aspect of cholinergic transmission, however, provides further evidence against a direct vasodilatory role for acetylcholine as a terminal transmitter in isolated cerebral blood vessels.

Identification of a peptide that protects the human acetylcholine receptor against antigenic modulation.

mAb 192 is a rat monoclonal antibody with very high affinity for the major immunogenic region (MIR) of the human muscle acetylcholine receptor (AChR). An epitope mimic of this antibody was selected from a phage display peptide library screened with mAb 192. The peptide-presenting phage has been shown to specifically bind to solid phase mAb 192 with an equilibrium dissociation constant (K(d)) of 8.45x10(-9) M, as directly measured with surface plasmon resonance. This value represents the avidity of the interaction between selected phage and mAb 192. A synthetic version of this peptide QPSPYNGWRMEI, referred to as MG15, binds to its selecting antibody and blocks the interaction of mAb 192 with human AChR. Peptide MG15 was able to protect acetylcholine receptors on human RD cells from antibody-mediated down-modulation. The negative charge of glutamic acid plays a important role in antibody binding. Replacement of the glutamic acid with an alanine completely abolishes the inhibitory activity.

Beta2-glycoprotein I-dependent anticardiolipin antibodies preferentially bind the amino terminal domain of beta2-glycoprotein I.

Many of the autoantibodies in antiphospholipid syndrome (APS) are directed against beta2-glycoprotein I (beta2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of beta2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed > or = threefold selectivity for beta2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti-beta2 antibodies are localized to the amino terminal domain of beta2-GPI.

Demonstration of a second pharmacologically active promoter region in the NGF gene that induces transcription at exon 3.

Nerve growth factor (NGF) has been demonstrated to facilitate neurite outgrowth, rescue neurons from injury, and prevent programmed cell death in neurons. However, the therapeutic potential of NGF is limited by metabolic instability and poor CNS penetration. These limitations might be circumvented by identifying compounds which increase endogenous production of NGF in the brain. We sought to determine the site of all pharmacologically inducible promoters in the NGF gene using a differential analysis based on semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Mouse L929 cells were serum deprived and NGF mRNA was induced by treatment with phorbol 12-myristate 13-acetate (PMA), 1,25-dihydroxy-vitamin D3 (calcitriol) or horse serum. An increase in transcripts initiating at exon 1 was noted in cDNA from cells induced with all three agents. In addition, we also observed an increase in cDNA transcripts that initiate at exon 3 and do not include exons 1 and 2 (4.38 +/- 0.42, 2.56 +/- 0.05 and 3.04 +/- 0.03 fold increase over control for PMA, calcitriol and serum, respectively). Each of these increases was completely inhibited in the presence of actinomycin D, indicating that the increased levels of mRNA were due to increases in transcription and not mRNA stabilization. These results confirm the previous demonstration of a promoter for NGF near exon 1 and establish a pharmacologically inducible promoter in the NGF gene near exon 3 that could be targeted for therapeutic intervention.

Apoptotic DNA fragmentation in the rat cerebral cortex induced by permanent middle cerebral artery occlusion.

Recent investigations have demonstrated internucleosomal DNA fragmentation in ischemic neuronal tissue. This type of fragmentation is characteristic of programmed cell death or apoptosis and suggests that neuronal death in stroke may be more complex than simple necrotic death. The present experiments provide a detailed examination of the regional localization and time course for apoptotic DNA fragmentation in the cerebral cortex following focal cerebral ischemia. Spontaneously hypertensive rats were subjected to permanent right middle cerebral artery occlusion and the cerebral cortices were examined for evidence of DNA fragmentation using electrophoretic, flow cytometric, and histological approaches. An electrophoretic examination of cortical DNA at 24 h after the occlusion indicated that the majority of nucleosomal ladders were in the transition zone or penumbra and the core of the infarction, with no fragmentation apparent in the contralateral normal cortex. A flow cytometric analysis of DNA fragmentation in intact cells revealed a similar pattern, with increased fragmentation observed in ischemic cortex vs. the contralateral cortex. Saggital sections taken 1.5 mm lateral to midline were collected from animals at 1, 4, and 24 h after the infarction and DNA fragmentation was examined histologically by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) staining. Quantitative analysis of these sections indicated that DNA fragmentation can be observed in the anterior and central area of the infarctions as soon as 1 h after the occlusion and that the extent and magnitude of the fragmentation increases at 4 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of apoptosis in acute neurodegenerative disorders.

Many toxic factors are generated during stroke that contribute directly to the death of neurons. Several recent studies suggest that a suicide-like phenomena similar to apoptosis or programmed cell death also contributes to the loss of neurons in stroke. The evidence implicating apoptosis in stroke can be divided into three categories; biochemical, molecular and pharmacological. Biochemical evidence: One hallmark of apoptosis is the early activation of destructive enzymes, including endonucleases and proteases. Endonuclease-mediated DNA fragmentation can be observed within 4 h after focal cerebral ischemia and precedes morphological evidence of cell death. Cells with damaged DNA appear to concentrate in the salvageable tissue of the penumbra while necrosis predominates in areas where the sustained lack of blood flow may make tissue salvage impossible. Molecular evidence: Bcl-2 is an anti-apoptotic gene that confers the ability to block apoptosis from a wide variety of stimuli. The levels of bcl-2 can be enhanced by viral gene delivery or transgenic methodology. In cortical tissue where bcl-2 was elevated, neurons were protected from a subsequent ischemic attack. In contrast to bcl-2, p53 is a pro-apoptotic protein. Levels of p53 are elevated after cerebral ischemia and transgenic p53 knockouts exhibit smaller infarcts than wild type control mice. Pharmacological evidence: The process of apoptosis typically involves the activation of enzymes and genes, leading to an irreversible committment to die. Inhibition of new protein synthesis by cycloheximide reduces brain damage after a stroke, suggesting that newly synthesized proteins are contributing to the death of neurons. In addition, inhibition of calpain (an enzyme implicated in certain forms of apoptosis) protects neurons in models of global ischemia, focal ischemia, and hypoxia. In conclusion, the observation that an apoptotic-like process contributes to stroke may have important therapeutic implications since therapies that inhibit apoptosis improve outcome in experimental stroke.

Identification of immunoreactive substance P in human and other mammalian endothelial cells.

Endothelial cells release both vasodilatory (e.g., PGI2, EDRF, oxygen radicals) and vasoconstrictor (e.g., EDCF) substances which modify vascular tone and contractility. We report the existence of the vasodilatory tachykinin substance P within endothelial cell scraping from human, rat and dog thoracic aorta and human pial arteries with values ranging from 1.0 +/- 0.1 (rat aorta) to 1.9 +/- 0.5 (dog aorta) fmol/mg protein. The immunoreactive component eluted with a retention time identical to that of radiolabelled substance P when analyzed by high performance liquid chromatography combined with radioimmunoassay. Cultured endothelial cells from bovine cerebral microvessels contained measurable levels of substance P in passages 3-8, suggesting the likelihood that these cells synthesize substance P. However, the level of gene expression must be low since efforts to demonstrate the presence of preprotachykinin mRNA by Northern blot analysis of dog and rat aortic endothelial cell RNA or by RNase protection analysis of rat aortic endothelial cell RNA was not successful.

The selectivity of MDL 74,721 in models of neurogenic versus vascular components of migraine.

MDL 74,721 (R)-2-(N1,N1-dipropylamino)-8-methylaminosulfonylmethyl-1,2,3,4-te trahydronaphthalene, a sulfonamidotetralin, has been found to exhibit a 10,000-fold greater potency in neurogenic versus vascular models of migraine. Sumatriptan, a relatively pure 5-HT1D/5-HT1B receptor agonist, also showed higher potency versus neurogenic inflammation. However, for sumatriptan the potency difference (100-fold) in the two pathophysiological models was less pronounced than seen for MDL 74,721. The affinity profile of MDL 74,721 at 5-HT1 receptor subtypes may in part explain its ability to differentiate these two physiological responses. MDL 74,721 demonstrated nanomolar affinity for 5-HT1A (12.7 +/- 0.3 nM) and 5-HT1D (41.3 +/- 10.9 nM) but considerably lower affinity for 5-HT1B receptors (> 1000 nM). Serotonin-like activity was seen in in vitro functional assays including inhibition of forskolin-stimulated cAMP accumulation in human 5-HT1D receptor-transfected fibroblasts or eliciting vasoconstriction in isolated human pial arteries. The intrinsic activity (relative to 5 - HT[E(Amax)]) and affinity (pD2) for the human cerebrovascular 5-HT receptors were: 5-HT (100%, 7.51 +/- 0.09), sumatriptan (94%, 6.85 +/- 0.1) and MDL 74,721 (66%, 5.70 +/- 0.23). In anaesthetised cats, treatment with MDL 74,721 resulted in a dose-related reduction in the percentage of carotid flow going through the arteriovenous anastomoses to the lungs, with an ED50 of 0.3 mg/kg i.v., the same as sumatriptan. However, in the guinea-pig neurogenic model, MDL 74,721 inhibited plasma protein extravasation with an ED50 of 0.023 microg/kg compared to 2.5 microg/kg for sumatriptan. MDL 74,721 was also effective in this model (in rats) after oral administration. In conclusion, MDL 74,721 demonstrates a preclinical profile consistent with anti-migraine efficacy. Its marked preference for inhibiting neurogenic inflammation makes this compound a useful tool for assessing the relative contribution of this pathophysiological mechanism to the human disease state.

Oxyhemoglobin inhibition of acetylcholinesterase activity.

The effects of human oxyhemoglobin (HbO2), human methemoglobin (MetHb), and porcine serum albumin (PSA) on the activity of acetylcholinesterase (AChE) isolated from Electrophorus electricus were examined. HbO2 produced a dose-dependent reduction in AChE activity. Fifty percent of activity was obtained at 5 microM HbO2, while 95% inhibition was obtained at 50 microM. In this concentration range MetHb and PSA had little effect on esterase activity.

Biostability and pharmacokinetics of LJP 920, an octameric Gal (alpha1-3) Gal conjugate for the inhibition of xenotransplantation rejection.

Antibodies to an alpha-galactosyl saccharide structure present in human serum are associated with hyperacute rejection and delayed xenograft rejection after pig-to-primate xenotransplantation. To overcome this major barrier to the xenotransplantation, LJP 920, a galactosyl alpha1-3 galactose (Gal (alpha1-3) Gal) coupled to a non-immunogenic platform at a valency of eight Gal (alpha1-3) Gal molecules/platform, was synthesized to clear circulating antibodies and to inhibit their production by B cells that produce these antibodies. Herein we report on the stability of UP 920 in biological media and its pharmacokinetic profile. Incubation of LJP 920 with mouse serum or liver microsomes at 37 degrees C for 2 days showed no indication of degradation of the conjugate as detected by a reversed-phase HPLC method, indicating that the conjugate is not subject to enzymatic metabolism. After intravenous administration of LJP 920 to mice at the doses of 20 and 100 mg kg(-1), UP 920 serum concentration decreased rapidly, showing a biphasic pattern, with a distribution half-life of 3 min and an elimination half-life of more than 30 min, respectively. The serum-to-erythrocyte concentration ratio of UP 920 was 33- and 36-fold excess at 0.5 and 5 min, respectively, after intravenous administration (100 mg kg(-1)). Both Cmax and AUC values increased in a dose-proportional manner. UP 920 displayed a great distribution to well-perfused tissues. It was eliminated mainly through renal excretion in the unchanged form, which accounted for 23% of the total amount within 8 h of dosing.

Analysis of neurogenic contractions induced by ML-1035 and other benzamides in the guinea-pig non-stimulated isolated ileum.

4-Amino-5-chloro-substituted benzamides have been shown to increase gastric motility in-vivo and enhance field-stimulated and peristaltic contractions in-vitro. The present experiments examined the contractile response to a series of benzamides in the guinea-pig non-stimulated ileum. Four benzamides elicited contractions in the isolated ileum which were expressed as a percentage of the contraction induced by 1 microM acetylcholine (% acetylcholine response = 12 +/- 2, 19 +/- 3, 26 +/- 2, 51 +/- 3, n = 13, 8, 17, and 21, with EC50 values of 0.85, 1.8, 5.7, and 14.2 microM for cisapride, zacopride, metoclopramide, and ML-1035 (4-amino-5-chloro-2-((2-methylsulphinyl)-ethoxy)-N- (2-(diethylamino)-ethyl)-benzamide hydrochloride), respectively). ML-1035 contractions were completely blocked by atropine and tetrodotoxin, while ganglionic blockade with hexamethonium was ineffective. Metoclopramide has been reported to sensitize postjunctional muscarinic receptors, however, ML-1035 did not enhance acetylcholine-induced contractions. Tropisetron (ICS 205-930, 1 microM), caused a parallel rightward shift in the concentration-response curve for both ML-1035 and zacopride (EC50 = 14.2 +/- 1.3 and 1.8 +/- 0.8 microM in the absence, and 26 +/- 2.7 and 6.9 +/- 2.3 microM in the presence of tropisetron for ML-1035 and zacopride, respectively) with apparent pKB values of 5.9 and 6.0 for the respective compounds. 5-Hydroxytryptaminergic receptor desensitization by 2-methyl-5-hydroxytryptamine (5-HT3) and 5-methoxytryptamine (5-HT4), attenuated the response to ML-1035.(ABSTRACT TRUNCATED AT 250 WORDS)

Pain mechanisms underlying vascular headaches. Progress Report 1989.

Vascular headaches are among the most prevalent yet poorly understood problems in clinical neurology. Headaches may develop in association with hypertension, seizures, stroke or without a recognizable pathophysiology such as during migraine and cluster headaches. Cephalic blood vessels (pial and dural vessels) are implicated as the most important source for all headaches and are innervated by sensory fibers which arise from ganglia innervating the forehead, scalp and neck. Sensory fibers contain vasoactive neuropeptides which become released from peripheral (perivascular) and central terminations to mediate vasodilation and pain, respectively. The presence of vascular headache implies activation of this final common pain pathway which we have termed the trigeminovascular system. The presence of vascular headache implies activation of this final common pain pathway which we have termed the trigeminovascular system. The existence of such a system a) clarifies certain pain patterns which develop following stimulation of cephalic blood vessels, b) suggests a mechanism to explain the referral of pain to the forehead, c) provides a mechanism to explain the action of certain antimigraine drugs, d) suggests a local mechanism which enhances blood flow under certain pathological conditions. Hence, this review will update existing knowledge about the trigeminovascular system and its role in headache pathophysiology.

Benefit of inhibiting SSAO in relapsing experimental autoimmune encephalomyelitis.

We have developed several series of potent and selective small molecule inhibitors of SSAO (AOC3/VAP-1) that also block trafficking of leukocytes to sites of inflammation. Blocking of SSAO-mediated leukocyte adhesion has recently been shown efficacious in several models of inflammatory diseases. We have examined the potential of SSAO inhibitors in neurological diseases, having previously demonstrated the efficacy of SSAO inhibition in a rat model of stroke. Here we show the effect of the small molecule SSAO inhibitor LJP 1207 (IC(50) human SSAO 17 nM; ratio IC(50) SSAO:MAO >5000), on relapsing-remitting experimental autoimmune encephalomyelitis (EAE), a mouse model that shares many characteristics with human multiple sclerosis. Clinical efficacy was observed when dosing with LJP 1207 was initiated either at the peak of initial flare or during remission. These data demonstrate the potential clinical benefit of small molecule anti-SSAO therapy in this model.

Effect of hemoglobin on sympathetic neurovascular transmission in the porcine cerebral circulation.

Subarachnoid hemorrhage (SAH) exposes the adventitia of the cerebral blood vessels to erythrocytes which lyse to release hemoglobin (Hb). Both SAH and Hb have been demonstrated to deplete adrenergic innervation and alter neurogenic responses in these vessels. We examined the effect of Hb on sympathetic adrenergic transmission in isolated pig cerebral arteries. The results indicate that Hb blocks neuronal [3H]norepinephrine ([3H]NE) uptake into anterior cerebral, internal carotid, and middle cerebral arteries in a dose-dependent manner. Gel-filtration experiments suggest that the mechanism underlying the [3H]NE uptake blockade involves binding of NE to Hb, rather than a specific blockade of the uptake pump. Although Hb blocked [3H]NE uptake, it did not influence electrically stimulated, tetrodotoxin (TTX)-sensitive [3H]NE release from cerebral arteries. These findings indicate that the presence of Hb in the subarachnoid space may alter cerebrovascular sympathetic transmission by preventing NE uptake. The altered neurogenic responses observed in the presence of Hb may be influenced by the affinity of NE for this protein but also may involve release of a substance other than NE from the sympathetic terminals.

Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: Cells die by one of two mechanisms, necrosis or programmed cell death. Necrosis has been implicated in stroke and occurs when the cytoplasmic membrane is compromised. Programmed cell death requires protein synthesis and often involves endonucleolytic cleavage of the cellular DNA. We assessed the potential contribution of programmed cell death to ischemia-induced neuronal death. METHODS: Cycloheximide (protein synthesis inhibitor; 1 mg/kg per 24 hours) or vehicle (1 mL/kg per 24 hours) was continuously infused into the right cerebral ventricle of spontaneously hypertensive rats. Neocortical focal ischemia was produced by tandem occlusion of the right common carotid artery and the ipsilateral middle cerebral artery. After 24 hours the brain was stained with 2% 2,3,5-triphenyltetrazolium and the ischemic zone quantitated. Protein synthesis was determined by [3H]methionine incorporation into acid-precipitated protein. DNA integrity was determined in isolated DNA by gel electrophoresis and in whole cells by flow cytometry. RESULTS: Continuous cycloheximide infusion caused approximately 70% reduction in cortical protein synthesis. Cycloheximide also reduced the size of the infarction produced by focal cerebral ischemia when compared with controls (ischemic brain volume, 147.5 +/- 25.9 and 188.7 +/- 16.8 mm3 for cycloheximide and saline, respectively; P < .01), suggesting that protein synthesis may contribute to cell death. Purified DNA from the ischemic zone showed evidence of endonucleolytic degradation when fractionated by gel electrophoresis. Flow cytometric analysis demonstrated increased propidium iodide fluorescence in intact cells isolated from ischemic cortex, indicating an increased accessibility of degraded DNA to the intercalating dye. CONCLUSIONS: New protein synthesis appears to contribute to ischemic cell death in which endonucleolytic DNA degradation is apparent. These observations implicate programmed cell death in ischemic injury and may open unique therapeutic approaches for the preservation of neurons in stroke.

Antibody affinities and relative titers in polyclonal populations: surface plasmon resonance analysis of anti-DNA antibodies.

This paper presents the equations and methodology for the measurement and interpretation of apparent dissociation constants for polyclonal populations of antibodies, where antigen is kept trace relative to antibody concentration. Surface plasmon resonance is used to determine K(d)s for the binding of anti-DNA antibodies to trace amounts of DNA antigen on a chip. Since the approach taken relies on equilibrium measurements, kinetic mass transport artifacts are avoided. The apparent K(d) is a weighted average of all the K(d)s for the clonally related subpopulations within the polyclonal pool, where each weighting factor is the relative titer (fractional presence) of the subpopulation. Titration curves appear as if there is one monoclonal population with that titer-weighted-average K(d). Implications of changes in the antibody affinity distribution within the population are discussed. The equations described herein provide a better physical understanding of the apparent K(d) that is obtained when a heterogeneous population of receptors is titrated against a trace ligand.

Subarachnoid blood and headache: altered trigeminal tachykinin gene expression.

Sensory axons from the trigeminal ganglion (V) innervate cephalic blood vessels and use the preprotachykinin gene products, substance P (SP) and neurokinin A (NKA), as putative neurotransmitters conveying nociceptive information. Blood in the subarachnoid space is accompanied by severe headache. We now report that this painful stimulus, which should enhance activity in V, specifically alters tachykinin peptide and mRNA levels in V and perivascular axons. Marked reductions in SP levels were observed in basilar artery segments within 4 hours after intracisternal blood injection which persisted for 48 hours and recovered by 7 days. SP peptide levels in V were elevated by 49% two days after blood injection. The changes in SP peptide levels were accompanied by increases in ganglionic content of the preprotachykinin mRNA that codes for the peptide. Blood-induced peptide depletion in arteries and subsequent increases in peptide and mRNA in V are consistent with increased neuronal activity and enhanced neuropeptide release. These results implicate the tachykinin-utilizing trigeminovascular neurons in the sequelae of subarachnoid hemorrhage.