Stereoselective pharmacokinetics of tetrahydropalmatine after oral administration of (-)-enantiomer and the racemate

Tetrahydropalmatine (THP) is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). THP is a racemic mixture which contains 50% of the (+) and 50% of (-) enantiomer. The (-) enantiomer accounts for most of the analgesic effects. Plasma concentrations of THP enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OJ column with quantification by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of rac-THP or (-)-THP. The pharmacokinetic profiles of the two enantiomers after dosing with rac-THP were significantly different both in rats and dogs. The mean C(max) and AUC(0-infinity) values in rats were 1.93 +/- 0.36 microg/ml and 6.65 +/- 2.34 microg x h/ml for the (-) enantiomer, and 1.11 +/- 0.25 microg/ml and 2.03 +/- 0.45 microg x h/ml for the (+) enantiomer. The mean C(max) and AUC(0-infinity) in dogs were 1.60 +/- 0.81 microg/ml and 9.88 +/- 2.58 microg x h/ml for the (-) enantiomer, while 0.36 +/- 0.21 microg/ml and 1.22 +/- 0.40 microg x h/ml for the (+) enantiomer. rac-THP at 40 mg/kg and (-)-THP at 20 mg/kg had very similar plasma concentration-time profiles, and C(max), AUC(0-infinity), and t(1/2) of the (-) enantiomer in both rats and dogs, indicating that the two treatments were equivalent with respect to the pharmacokinetic properties of the (-) enantiomer.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

Pharmacological identification of the major subtypes of adrenoceptors involved in the canine external carotid vasoconstrictor effects of adrenaline and noradrenaline.

This study investigated the potential effects of adrenaline and noradrenaline on the external carotid blood flow of vagosympathectomised dogs and the receptor mechanisms involved. One minute (1 min) intracarotid infusions of adrenaline and noradrenaline produced dose-dependent decreases in external carotid blood flow without changes in blood pressure or heart rate. These responses, which remained unaffected after saline, were: (i) mimicked by the adrenoceptor agonists, phenylephrine (alpha1) and BHT933 (6-Ethyl-5,6,7,8-tetrahydro-4H-oxazolo [4,5-d] azepin-2-amine dihydrochloride; alpha2); (ii) abolished after phentolamine (2000 microg/kg) unmasking a vasodilator component (subsequently blocked by propranolol; 1000 microg/kg); and (iii) partly blocked by rauwolscine (30 and 100 microg/kg), and subsequently abolished by prazosin (100 microg/kg). Accordingly, rauwolscine (100 and 300 microg/kg) markedly blocked the responses to BHT933 without affecting those to phenylephrine; likewise, prazosin (100 microg/kg) markedly blocked the responses to phenylephrine without affecting those to BHT933. These results show that both alpha1- and alpha2-adrenoceptors mediate vasoconstriction within the canine external carotid circulation. Moreover, after blockade of alpha1/alpha2-adrenoceptors, both adrenaline and noradrenaline exhibit a beta-adrenoceptor-mediated vasodilator component.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

Homoepiboxidines: further potent agonists for nicotinic receptors

Homoepiboxidine (3) and the corresponding N-methyl (4) and N-benzyl (5) derivatives were prepared from a 6beta-carbomethoxynortropane (8). Affinities and functional activities at neuromuscular, central neuronal and ganglionic-type nicotinic receptors were compared to those of epibatidine 1, and epiboxidine 2. Homoepiboxidine had equivalent affinity/activity to epiboxidine at neuromuscular, neuronal alpha4beta2, and most alpha3-containing ganglionic-type nicotinic receptors. The N-substituted derivatives showed reduced affinity/activity at most receptor subtypes. Replacement of the methylisoxazole moiety of 3 and 4 with a methyloxadiazole moiety provided analogues 6 and 7, which had greatly reduced affinity/activity in virtually all assays at nicotinic receptors. Marked analgetic activity in mice occurred at the following ip doses: epibatidine 10 microg/kg; epiboxidine 25 microg/kg; homoepiboxidine 100 microg/kg; N-methylhomoepiboxidine 100 microg/kg; the methyloxadiazole (6) 100 microg/kg. The time course at such ip doses was significantly longer for homoepiboxidine 3 with marked analgesia still manifest at 30 min post-injection. Epiboxidine and the homoepiboxidines were less toxic than epibatidine.     

Stimulation of brain mast cells by compound 48/80, a histamine liberator, evokes renin and vasopressin release in dogs

Because degranulation of brain mast cells activates adrenocortical secretion (41, 42), we examined whether activation of such cells increases renin and vasopressin (antidiuretic hormone: ADH) secretion. For this, we administered compound 48/80 (C48/80), which liberates histamine from mast cells, to pentobarbital-anesthetized dogs. An infusion of 37.5 microg/kg C48/80 into the cerebral third ventricle evoked increases in plasma renin activity (PRA), and in plasma epinephrine (Epi) and ADH concentrations. Ketotifen (mast cell-stabilizing drug; given orally for 1 wk before the experiment) significantly reduced the C48/80-induced increases in PRA, Epi, and ADH. Resection of the bilateral splanchnic nerves (SPX) below the diaphragm completely prevented the C48/80-induced increases in PRA and Epi, but potentiated the C48/80-induced increase in ADH and elevated the plasma Epi level before and after C48/80 challenge. No significant changes in mean arterial blood pressure, heart rate, concentrations of plasma electrolytes (Na+, K+, and Cl-), or plasma osmolality were observed after C48/80 challenge in dogs with or without SPX. Pyrilamine maleate (H1 histaminergic-receptor antagonist) significantly reduced the C48/80-induced increase in PRA when given intracerebroventricularly, but not when given intravenously. In contrast, metiamide (H2 histaminergic-receptor antagonist) given intracerebroventricularly significantly potentiated the C48/80-induced PRA increase. A small dose of histamine (5 microg/kg) administered intracerebroventricularly increased PRA twofold and ADH fourfold (vs. their basal level). These results suggest that in dogs, endogenous histamine liberated from brain mast cells may increase renin and Epi secretion (via the sympathetic outflow) and ADH secretion (via the central nervous system).     

Systemic efficacy of nodulisporic acid against fleas on dogs

Nodulisporic acid A (NSA) is a novel natural product from a new structural class that was shown previously to have insecticidal activity against blowfly larvae. To determine if there was useful systemic efficacy against fleas (Ctenocephalides felis). NSA was evaluated in an artificial membrane flea feeding device and in dogs. In the artificial membrane flea feeding device, adult C. felis were allowed to feed on bovine blood containing various concentrations of NSA through a Parafilm membrane. NSA killed the fleas with a 50% lethal concentration of 0.68 microg/ml and was approximately 10-fold more potent than the systemic insecticide ivermectin. In the initial probe dog test, a single beagle was challenged with 100 C. felis before oral dosing with 15 mg/kg of NSA. Flea counts conducted at 72 hr postdosing showed an 88% reduction relative to control. Re-challenge of the same dog at 5 days postdosing showed 50% reduction of fleas at day 7, demonstrating some residual flea activity. In a confirmatory study, 8 dogs were challenged with 100 fleas just before oral dosing with 15 mg/kg of NSA (4 dogs) or vehicle (4 dogs). There was 99% reduction of fleas at 48 hr postdosing in the NSA-treated dogs relative to control. Additional challenges with 100 fleas were performed on these 8 dogs at 48-hr intervals to determine the duration of efficacy, and there was 97, 51, and 0% reduction of fleas relative to control on days 4, 6, and 8, respectively. No adverse effects were observed in the dogs in these studies. These data show that NSA has potent oral activity in the dog for the control of fleas, while lacking overt mammalian toxicity.     

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.     

Effects of a GnRH cytotoxin on reproductive function in peripubertal male dogs

Methods for long-term or permanent disruption of reproductive function via nonsurgical techniques are needed for a variety of species, including companion animals. In a previous study, we demonstrated the ability of a cytotoxin (pokeweed antiviral protein-PAP) conjugated to d-Lys(6)-GnRH, to disrupt reproductive function in adult male dogs. The objective of the present study was to examine the ability of a d-Lys(6)-GnRH-PAP conjugate to disrupt reproductive function in peripubertal male dogs. Peripubertal male dogs (n=15; approximately 16-32 weeks old) were treated with d-Lys(6)-GnRH-PAP as follows: dogs (n=7; Group I) received GnRH-PAP (0.1 mg/kg SQ) with a second treatment (0.25 mg/kg) 20 weeks later. An additional group (n=3; Group II) of peripubertal dogs was treated with GnRH-PAP (0.25 mg/kg) twice (20 weeks apart). Control dogs (n=5) received d-Lys(6)-GnRH analog (0.0045 mg/kg SQ) without PAP. Efficacy was assessed by monitoring testis size, serum concentrations of testosterone and LH, as well as LH release subsequent to a GnRH (5 microg/kg) stimulus. Dogs in Group I (n=5) that did not respond to the initial two treatments were given a third GnRH-PAP injection (0.25 mg/kg), 12 months after the initial treatment. The initial GnRH-PAP treatment in peripubertal male dogs did not affect testis growth, LH release or serum testosterone concentrations; however, administration of a higher dose of GnRH-PAP after puberty resulted in a marked and rapid decline in testis size, serum testosterone concentrations and LH responsiveness to GnRH stimulation in 9 of 10 dogs. Suppression of reproductive function was maintained in treated dogs for 18-50 weeks; four dogs had suppression of reproductive activity through the end of the study. In conclusion, GnRH-PAP given after puberty markedly suppressed reproductive activity. Due to variability in the response and duration of suppression after treatment with GnRH-PAP, more research is required to determine its efficacy for nonsurgical sterilization of the male dog.     

The spleen modulates the febrile response of guinea pigs to LPS

The febrile responses of splenectomized (Splex) or sham-operated (Sham) guinea pigs challenged intravenously or intraperitoneally with lipopolysaccharide (LPS) 7 and 30 days after surgery were evaluated. FITC-LPS uptake by Kupffer cells (KC) was additionally assessed 15, 30, and 60 min after injection. LPS at 0.05 microg/kg iv did not evoke fever in Sham animals but caused a 1.2 degrees C core temperature (T(c)) rise in the Splex animals. LPS at 2 microg/kg iv induced a 1.8 degrees C greater T(c) rise of the Splex animals than of their controls. LPS at 2 and 8 microg/kg ip 7 days postsurgery induced 1.4 and 1.8 degrees C higher fevers, respectively, in the Splex than Sham animals. LPS at 2 and 8 microg/kg ip 30 days postsurgery also increased the febrile responses of the asplenic animals by 1.6 and 1.8 degrees C, respectively. FITC-LPS at 7 days was detected in the controls within KC 15 min after its administration; the label density was reduced at 30 min and almost 0 at 60 min. In the Splex group, in contrast, the labeling was significantly denser and remained unchanged through all three time points; this effect was still present 30 days after surgery. Similar results were obtained at 60 min after FITC-LPS intraperitoneal injection. Gadolinium chloride pretreatment (-3 days) of the Splex group significantly reduced both their febrile responses to LPS (8 microg/kg ip) and their KC uptake of FITC-LPS 7 days postsurgery. Thus splenectomy increases the magnitude of the febrile response of guinea pigs and the uptake of systemically administered LPS.     

Effect of ivermectin on the cellular and humoral immune responses of rabbits

The objective of this paper is to determine the effect of ivermectin administration on cell mediated (CMI) and humoral immunity (HI) of rabbits. CMI against dinitrochlorobenzene (DNCB) and sheep red blood cells (SRBC) in rabbits was determined by delayed-type hypersensitivity and macrophage engulfment assay (MEA), respectively; whereas, HI to Pasteurella multocida B2 vaccine and SRBC was determined by indirect haemagglutination assay (IHA) and Jerne hemolytic plaque formation assay (JHPFA), respectively. The rabbits were divided into four major groups (A through D) each subdivided into four sub-groups (1 through 4). Rabbits of group A served as vehicle control while those of groups B, C and D were treated with ivermectin at the dose rates of 200 microg/kg, 400 microg/kg and 600 microg/kg b.w., respectively. Cellular immunity was determined in sub-groups 1 and 2 through DNCB and MEA, respectively while HI was determined in sub-groups 3 and 4 through IHA and JHPFA, respectively. The skin sensitivity to DNCB at 24 and 48 h and macrophage engulfment of SRBC were highest (P>0.05) in rabbits administered with 600 microg/kg b.w. The highest geometric mean titers (14.00+/-0.31) and number of plaque forming units (1860+/-0.75) were found in rabbits that received ivermectin at a dose of 600 microg/kg b.w. followed, in order by the groups that received 400 microg/kg, 200 microg/kg b.w. and controls. Leukocyte counts were significantly higher in ivermectin-treated groups (C and D) than group A (vehicle control) and B (ivermectin at the rate of 200 microg/kg). A graded dose immune response suggested an immunopotentiating effect of ivermectin at higher doses.     

Changes in serum luteinizing hormone (LH) concentrations in response to luteinizing hormone releasing hormone (LHRH) in bull calves that attained puberty early or late

The objectives of this study were to determine if the response to luteinizing hormone releasing hormone (LHRH) could be used to select bull calves capable of early sexual maturation and to establish the optimum route and dose of LHRH. In Trial 1, at 4, 10 and 20 week of age, 20 calves were treated iv with 2 microg/kg body weight of LHRH 1 and 5h after commencing a 9-h period of blood sampling. Bulls were separated into early and late maturing (n=10), based on age at puberty (scrotal circumference (SC) of >or=28 cm). At 4 and 20 week of age, peak serum LH concentrations and area under the LH response curve in response to LHRH were lower (P<0.05) in early- versus late-maturing bulls. In Trial 2, calves at 20 week of age were given LHRH as follows: 2 microg/kg body weight iv (n=6), im (n=6) or sc (n=6); 5 microg/kg im (n=6), or ischio-rectally (ir, n=6) or sc (n=6); and 10 microg/kg im (n=6) or sc (n=6). Serum LH concentrations were at a plateau from 30 to 165 min after treatment with 5 microg/kg of LHRH (im or ir; P>0.05). We concluded that the LH responses to LHRH in calves at 4 and 20 week of age could facilitate the development of a simple test (one blood sample prior to treatment with LHRH and a second during the period of sustained response to LHRH) to select early-maturing bulls.     

The haemodynamic and catecholamine response to xenon/remifentanil anaesthesia in Beagle dogs

The noble gas xenon seems to have minimal cardiovascular side-effects and so may be an ideal anaesthetic agent when investigating cardiovascular physiology. In comparison with standard modern anaesthetics, we investigated the haemodynamic and hormonal effects of xenon in Beagle dogs. After a 30 min baseline period, anaesthesia was induced with propofol and maintained with either (1) 1.2% isoflurane/70% nitrous oxide (N(2)O), (2) 0.8% isoflurane/0.5 microg/kg/min remifentanil or (3) 63% xenon/0.5 microg/kg/min remifentanil (n = 6 per group). Haemodynamics were recorded and blood samples taken before and 60 min after induction. Mean arterial blood pressure (MAP) was higher in conscious dogs than during isoflurane/N(2)O (86 +/- 2 vs. 65 +/- 2 mmHg, mean +/- SEM) and isoflurane/remifentanil anaesthesia (95 +/- 2 vs. 67 +/- 3 mmHg), whereas MAP did not decrease significantly in response to xenon/remifentanil anaesthesia (96 +/- 4 vs. 85 +/- 6 mmHg). Bradycardia was present during isoflurane/remifentanil (54 +/- 2/min) and xenon/remifentanil (40 +/- 3/min), but not during isoflurane/N(2)O anaesthesia (98 +/- 3/min, P < 0.05). Xenon/remifentanil anaesthesia induced the highest reduction in cardiac output (CO) (-61%), and the highest increase in systemic vascular resistance (+120%) among all treatment groups (P < 0.05). A simultaneous increase in endogenous adrenaline and noradrenaline concentrations could only be observed in the xenon/remifentanil group, whereas angiotensin II and vasopressin concentrations increased in all groups. In conclusion, xenon/remifentanil anaesthesia maintains MAP but reduces heart rate and CO and is associated with a considerable stimulation of vasopressor hormones in Beagle dogs. Therefore, xenon/remifentanil exerts a new quality of adverse haemodynamic effects different from volatile anaesthetics and may not perform better during studies of cardiovascular physiology.     

Pyrexia, anorexia, adipsia, and depressed motor activity in rats during systemic inflammation induced by the Toll-like receptors-2 and -6 agonists MALP-2 and FSL-1

Macrophage-activating lipopeptide-2 (MALP-2) from Mycoplasma fermentans has been identified as a pathogen-associated molecular pattern of Mycoplasmas that causes activation of the innate immune system through the activation of the heterodimeric Toll-like receptors (TLRs)-2 and -6. The aim of this study was to characterize the ability of MALP-2 and a synthetic analog fibroblast-stimulating lipopeptide-1 (FSL-1; represents the NH2-terminal sequence of a lipoprotein from M. salivarium) to act as exogenous pyrogens, to induce formation of cytokines (endogenous pyrogens), and to cause sickness behavior, such as depressed motor activity, anorexia, and adipsia. For this purpose, body temperature, activity, food intake, and water intake were recorded for 3 days by use of telemetry devices in several groups of rats treated with MALP-2/FSL-1 or the respective control solutions. Intraperitoneal injections of FSL-1 caused fever at doses of 10 or 100 microg/kg, which was preceded by a pronounced phase of hypothermia in response to a dose of 1,000 microg/kg. The maximal fever (a peak of 1.5 degrees C above baseline) was caused by the 100 microg/kg dose with almost identical responses to both MALP-2 and FSL-1. Fever was accompanied by pronounced rises of the proinflammatory cytokines TNF and IL-6 in plasma. Treatment with the TLR-2 and -6 agonists further induced a dose-dependent manifestation of anorexia and adipsia, as well as a reduction of motor activity. We could thus demonstrate that activation of TLR-2 and -6 can induce systemic inflammation in rats accompanied by the classical signs of brain-controlled illness responses.     

Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T(b)) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic d-Phe-Cys-Try-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP; 0.1-1.0 microg) reduced fever induced by LPS (5.0 microg/kg) but did not change T(b) at ambient temperatures of either 20 degrees C or 28 degrees C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0-10.0 mg/kg, 3.0-30.0 microg, and 1-100 ng, respectively) produced a dose-dependent increase in T(b). Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 microg icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 microg), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2alpha) (500.0 ng) but not the fever induced by IL-1beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE(2) levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.     

Dissociation of H-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2b/H-2b). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (all H-2b/H-2b), FY7 failed to induce the primary in vitro generation of anti-H-2b CTL by (B10.A x A)F1 (H-2a/H-2a) or B10.D2 x BALB/c)F1 (H-2d/H-2d) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2b CTL. Quantitative absorption tests with H-2Kb and H-2Db antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2Kb product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2Kb CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2Kb-gene product expressed by FY7.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

Biochemical studies on the H-2K antigens of the MHC mutant bml

Biochemical analysis of the H-2K-gene product from the MHC mutant strain bml and from the C57BL/6 parent strain has been carried out in order to characterize the structural differences between parent and mutant K-gene products. Based on comparative tryptic peptide mapping of the cyanogen bromide fragments from these glycoproteins, two peptide differences were localized to the CN-Ia fragment. Partial amino-acid sequence analysis revealed two alterations in the primary structure of Kbml involving substitutions of tyrosine for arginine at position 155, and tyrosine for leucine at position 156. Both of these amino-acid replacements require a minimum of two nucleotide base changes at the nucleic acid level. These changes were the only alterations noted differentiating the Kbml and Kb glycoproteins. However, because our techniques allow us to analyze only 75 to 80 percent of the extra cellular portion of H-2Kb, it is possible there are other undetected changes. Nonetheless, the biochemical data are consistent with the hypothesis that the structural alterations noted in the Kbml mutant glycoprotein are directly related to the observed immunological specificity relative to the parent Kb molecule. Peptide comparisons of the Kb molecules of two C57BL/6 sublines and of the H-2b lymphoblastoid cell line, EL-4, disclosed no difference.     

Effects of medium containing heparin and theophylline on capacitation and metabolic enzyme activities of ejaculated spermatozoa from dogs with asthenozoospermia

The percentages of motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR) of four beagle dogs with asthenozoospermia (AS) and five normal beagle dogs were determined during 7 h of incubation. The metabolic enzyme activities of the sperm was examined after 0 and 4 h of incubation. The sperm were incubated in canine capacitation medium (CCM) and CCM containing either 20 microg ml(-1) heparin (HE), 10 microg ml(-1) theophylline (TH) or 20 microg ml(-1) HE + 10 microg ml(-1) TH in glass tubes at 38 degrees C under 5% CO2 in air. The %HA and %AR were determined by counting the sperm exhibiting star-spin like movement and by the triple stain technique. The spermatozoa in HE + TH CCM were homogenized and centrifuged, and the metabolic activities of hexokinase, fructokinase, glucose-6-phosphodehydrogenase (G6PD), and pyruvate kinase in the sperm cytosol in the supernatant was measured with a spectrophotometer. The mean %MO and %HA values of both AS and normal dogs in the four types of CCM were highest in HE + TH CCM, with a mean %HA in HE + TH CCM of 78 +/- 5% (S.E.) after 7 h of incubation. However, there was little difference in %AR among the four types of CCM. The mean activities of the four enzymes in the sperm of AS dogs before incubation was significantly lower than in the sperm of normal dogs (P < 0.05, 0.01). However, after 4 h of incubation the activities of all enzymes in the sperm of both AS and normal dogs was clearly higher in HE + TH CCM than in the control CCM. These findings indicate that HE and TH in the medium are effective inactivating metabolic enzymes, maintaining longer sperm motility, and efficiently inducing HA even of the sperm of AS dogs.