Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.

Interleukin-1 in human ovarian cells and in peripheral blood monocytes increases during the luteal phase: evidence for a midcycle surge in the human.

OBJECTIVE: Resident ovarian macrophages are thought to be critically involved in cyclic ovarian events. A prominent macrophage product, interleukin-1, has been shown to affect ovarian cell function. In this report we present evidence for an intrafollicular periovulatory interleukin-1 surge. Additionally, we demonstrate that interleukin-1 beta messenger ribonucleic acid in peripheral blood monocytes increases threefold during the luteal phase of the menstrual cycle over that found in the follicular phase. STUDY DESIGN: Follicular fluid cells isolated as a byproduct of transvaginal oocyte retrievals in gonadotropin-stimulated in vitro fertilization cycles were immunoprobed for the presence of interleukin-1 protein. Late follicular phase cumulus and granulosa cells obtained from an aspirated preovulatory follicle were likewise probed. RESULTS: Although the in vitro fertilization-retrieved cells stained positive for interleukin-1 protein, the late follicular phase cells were devoid of the protein. Granulosa cells from in vitro fertilization cycles were examined for interleukin-1 protein binding sites with iodinated interleukin-1 alpha protein. These cells were found to have approximately 2000 binding sites per cell. Poly A+ messenger ribonucleic acid isolated from peripheral blood monocyte samples from women during the follicular and luteal phases and from male controls were probed for interleukin-1 ribonucleic acid content by means of Northern analysis. The luteal samples contained a threefold higher interleukin-1 messenger ribonucleic acid content that did the follicular phase samples or the controls. CONCLUSION: The ovarian interleukin-1 protein increase taken together with increased peripheral blood monocyte interleukin-1 messenger ribonucleic acid suggests that interleukin-1 production increases at midcycle.

Evidence for the presence of the estrogen receptor in the ovary of the baboon (Papio anubis).

Although intraovarian estrogen has been firmly established as an important factor in the regulation of ovarian follicular development and function in the rat, an autocrine-paracrine role for estrogen in the primate ovary is not yet established. Immunocytochemical identification of an estrogen receptor in the monkey follicle was negative, but it was positive for the granulosa cells of antral follicles of the human ovary. In the present study baboon ovaries obtained during the follicular phase were examined for the presence of estrogen receptor by immunocytochemical analysis of frozen sections and Northern blot analysis of RNA extracts of the ovaries. Immunocytochemistry identified the estrogen receptor in the granulosa cells of healthy appearing and atretic or cystic-like antral follicles and in occasional, but rare, thecal cells. The ovaries contained a prominent mRNA species for the estrogen receptor, approximately 7 kilobases in size, which was present in relatively low abundance compared to that in the nonpregnant baboon uterus, but in a similar abundance to the estrogen receptor mRNA content of the pregnant endometrium. These studies are the first to report the presence of estrogen receptor mRNA in the ovary of a primate. These results in conjunction with the immunocytochemical studies firmly establish the presence of the estrogen receptor in the primate ovary and suggest an autocrine-paracrine role for intraovarian estrogen in primate ovarian physiology.

Ovarian steroids modulate human monocyte tumor necrosis factor alpha messenger ribonucleic acid levels in cultured human peripheral monocytes.

OBJECTIVE: To determine whether tumor necrosis factor alpha (TNF-alpha) messenger ribonucleic acid (mRNA) levels in human peripheral monocytes are regulated by ovarian steroids. DESIGN: Human granulosa-luteal cells and cultured, activated human peripheral monocytes were subjected to Northern blot analysis for TNF-alpha mRNA. SETTING: Academic research laboratory. PATIENTS: Two human female volunteers of reproductive age and in the luteal phase of the menstrual cycle. RESULTS: Human granulosa-luteal cells produce TNF-alpha mRNA. Physiological levels of progesterone (P) and estradiol (E2) modulate TNF-alpha mRNA from peripheral blood monocytes with an apparent inverse relationship between steroid concentration and TNF-alpha message. CONCLUSIONS: Progesterone and E2 at physiological concentrations regulate TNF-alpha mRNA production. The P antagonist mifepristone (RU486) and the E2 agonist/antagonist tamoxifen modulate total TNF-alpha mRNA levels, suggesting involvement of specific receptors.

Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist.

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.

Production of interleukin 2 and interleukin 4 by immune CD4-CD8+ and their role in the generation of antigen-specific cytotoxic T cells.

In this report we investigated the production and role of interleukin (IL)2 and IL4 in the generation of antigen-specific cytotoxic T cells (CTL). We used as our model the ultraviolet-light-induced epithelial tumor 1591, a highly immunogenic regressor tumor which evokes a strong cell-mediated immune response leading to rejection. We show that IL2 and IL4 are differentially required for the development of optimal cytolytic activity to the 1591 tumor in primary and secondary in vitro splenic cultures. First, anti-IL2 receptor monoclonal antibody (mAb) significantly decreased specific cytotoxicity in both primary and secondary splenic mixed lymphocyte-tumor cell culture (MLTC) cultures, but anti-IL4 mAb inhibited the cytotoxic responses only secondary and not primary cultures. Second, when supernatants from MLTC were tested for lymphokine activity, primary cultures produced only IL2 while secondary cultures produced both IL2 and IL4. Splenic cells were then depleted of CD4+ cells by negative selection, or enriched for CD8+ cells by positive selection, and tested for lymphokine production and requirements. CD8+ cells could not generate significant CTL activity in primary cultures, but could in secondary MLTC. The addition of mAb to either IL2 or IL4 significantly inhibited the generation of CTL by CD8+ cells in these secondary MLTC.CD8+ cells were also found to produce both IL2 and IL4 in secondary MLTC by functional and Northern blot analysis. The production of IL2 and IL4 by CD8+ cells occurs during different phases of culture, with IL2 being produced early (days 1 and 2) and IL4 late (days 3-5). In addition, the requirement of CD8+ cells for both IL2 and IL4 is unique for that lymphokine. These results suggest that both IL2 and IL4 are both produced and required by CD8+ cells during secondary MLTC, and suggest an additional cellular source of IL4 production besides CD4+ T cells during antigen-specific CTL responses.

Human follicular fluids contain tissue macrophages.

Tissue cells obtained from the follicular fluids of women undergoing in vitro fertilization and embryo transfer were found to contain interleukin-1 beta mRNA by Northern analysis. Since macrophages are known to produce interleukin-1 beta, we examined the follicular fluids of 20 women undergoing in vitro fertilization as well as tissue sections of normal human ovary for the presence of macrophages and monocytes. Although granulosa-luteal cells predominate in follicular fluid, we found that resident macrophages and monocytes comprise 5-15% of human follicular tissue cells. In addition, we observed that macrophages are present in the human ovarian follicle as well as in the corpus luteum.

Cultured human luteal peripheral monocytes secrete increased levels of interleukin-1.

Interleukin-1 (IL-1), an immune monokine secreted by activated macrophages and monocytes, appears to be intimately involved in endocrine phenomena. It is well established that IL-1 mediates a number of immune and inflammatory responses. In addition, the production of IL-1 appears to be modulated by the gonadal estradiol and progesterone, leading to this study of IL-1 secretion by cultured peripheral monocytes isolated at varying times during the menstrual cycle and pregnancy. Circulating peripheral monocytes were isolated from eight women undergoing human menopausal gonadotropin-stimulated ovulation induction during the late follicular phase just before hCG administration and from eight similarly stimulated women on the 12th day after hCG administration. Peripheral monocytes were also isolated from six women during both second and third trimester pregnancy. After 48 h in culture, conditioned media were assayed for IL-1 bioactivity using the D1O lymphocyte proliferation assay. Significantly more IL-1 bioactivity was secreted by monocytes isolated in the luteal phase of the cycle (52.4 +/- 17.5 IU/mL) compared to that in late follicular phase (5.2 +/- 0.9 IU/mL; P = 0.001) or third trimester of pregnancy (7.0 +/- 1.5 IU/mL; P = 0.006). Thus, peripheral monocyte IL-1 secretion appears to be increased by luteal levels of progesterone, although further elevation of progesterone during pregnancy returns IL-1 levels to the preovulatory baseline. In addition, basal body temperatures obtained in four women during third trimester pregnancy were 98 F or less. Thus, IL-1 secretion from cultured monocytes appears to increase with luteal concentrations of progesterone and decrease to preovulatory levels at higher concentrations of the steroid during pregnancy, which may account for the dissociation of high progesterone levels and elevated basal body temperature during late pregnancy.

Progesterone and estradiol modulate interleukin-1 beta messenger ribonucleic acid levels in cultured human peripheral monocytes.

The relationship between the endocrine system and immune monokines, such as interleukin-1 (IL-1), is of increasing interest. IL-1, a protein secreted by peripheral monocytes and tissue macrophages, mediates a wide variety of immune responses, and its production appears to be inversely related to the level of gonadal steroids. In this report, we have investigated the relationship between estradiol and progesterone concentrations and the level of IL-1 beta mRNA in cultured human peripheral monocytes and pelvic macrophages. Human peripheral monocytes, isolated during the luteal phase of the menstrual cycle, were activated with lipopolysaccharide (10 micrograms/mL). Cellular RNA was isolated and analyzed by Northern analysis using an 800-basepair IL-1 beta cDNA probe. Hybridization with 32P-labeled probe showed maximal levels of IL-1 beta mRNA occurring between 3 and 7 h of culture. Cultures of lipopolysaccharide-activated human peripheral monocytes incubated for 3-6 h with increasing amounts of progesterone or estradiol (0-10(-5) M) in the presence of either 5% fetal calf serum or 0.1% BSA demonstrated an inverse relationship between IL-1 beta mRNA levels and steroid concentration. In both cases, IL-1 beta mRNA levels decreased by 80-90% as the progesterone concentration increased to 10(-5) M and by 70-90% as the estradiol concentration increased similarly. A similar 80% decrease in IL-1 beta mRNA was observed with peritoneal macrophages incubated with increasing amounts of progesterone. This reciprocal relationship between IL-1 beta mRNA and gonadal steroids may have important ramifications in reproductive biology for both embryonic implantation and fetal survival as well as for clinically relevant changes in bone mass.

A comparison of the cellular electrophysiology of mexiletine and sotalol, singly and combined, in canine Purkinje fibers.

To determine if combinations of mexiletine and sotalol retain Class I and III electrophysiologic actions, using standard microelectrode techniques, we examined the electrophysiologic effects on canine Purkinje fibers of solutions of mexiletine (3.1-100 microM), sotalol (3.1-400 microM), and combinations of the two drugs, at stimulation frequencies of 1.5 and 2.0 Hz. The combinations consisted of 12.5 microM of one drug combined with 3.1, 12.5, and 50 microM of the other. Mexiletine caused a concentration dependent depression of Vmax, the degree of depression always being greater at the more rapid frequency. At concentrations above 50 microM, sotalol depressed Vmax slightly. Increasing the stimulation frequency did not result in further Vmax depression. The addition of sotalol did not alter the Vmax depression produced by mexiletine but prior exposure to sotalol attenuated the effect of subsequent mexiletine. Both drugs reduced action potential amplitude and in combination their effects on this parameter were additive. Sotalol prolonged and mexiletine shortened action potential duration. Low concentrations of mexiletine reversed the prolongation caused by sotalol, whereas the addition of sotalol did not alter the effect of mexiletine. Mexiletine shortened the effective refractory period at low concentrations and prolonged it at high concentrations. Sotalol prolonged the effective refractory period at all concentrations. Exposure to a low concentration of sotalol did not alter the effects on the effective refractory period of subsequent exposure to mexiletine but a low concentration of mexiletine reduced the prolongation from subsequent sotalol. Thus, the combination of mexiletine and sotalol may add a Class II action to the Class I effects of mexiletine but the mexiletine prevents the Class III effects of sotalol.

The cellular electropharmacology of the simultaneous administration of propranolol and mexiletine: a class I and II antiarrhythmic drug combination.

The addition of propranolol to mexiletine may reduce the adverse effects of mexiletine and possibly increase its efficacy. We compared the cellular electrophysiologic effects of this combination with mexiletine and propranolol alone, over concentration ranges of 3.2 to 100 microM mexiletine and 0.80 to 25.0 microM propranolol, using standard microelectrode techniques and a stimulation frequency of 1.5 Hz. Mexiletine and propranolol both depressed the rate of rise of phase 0 (Vmax) with concentration-response curves of similar slope and a relative potency of 8:1, propranolol to mexiletine. Thus, combinations of 8:1 molar ratios of mexiletine to propranolol were assessed. The combination depressed Vmax to the same extent as mexiletine or propranolol alone. Mexiletine, propranolol and the combination all shortened action potential duration (APD) to the same extent. At low concentration mexiletine, propranolol and the combination shortened effective refractory period (ERP). At 25 microM mexiletine, this effect reversed and ERP began lengthening. The effect of the combination paralleled mexiletine while with propranolol alone the reversal did not occur until the highest concentration was reached. Mexiletine prolonged ERP relative to APD, an effect not shared by propranolol and attenuated with the combination (P less than 0.05). We conclude that combining propranolol with mexiletine does not alter any of the cellular electrophysiologic effects of mexiletine except the prolongation of ERP relative to APD. Although this may be an important antiarrhythmic effect, the extent by which in vivo concentrations of propranolol may reduce the clinical antiarrhythmic efficacy of mexiletine is likely to be negligible.

Persistence of the electrophysiologic effects of mexiletine in canine Purkinje fibers alters the response to subsequent hypoxia.

The persistence of cellular electropharmacologic effects of mexiletine on canine Purkinje fibers was studied utilizing standard microelectrode techniques and two different protocols. In the first, the tissue was exposed to hypoxic perfusion before and 30 min after perfusion with one of the following: mexiletine hydrochloride 6.25 microM solution, mexiletine hydrochloride 12.5 microM solution, or drug-free Tyrode's solution. With the higher concentration of mexiletine, depression of the maximal upstroke velocity (Vmax) persisted 30 min after drug washout and subsequent exposure to hypoxia did not result in the anticipated shortening of action potential duration but did prevent the restoration of normal Vmax. After perfusion with the lower concentration of mexiletine, Vmax was not depressed and hypoxic action potential duration shortening was not prevented. In the second protocol, Purkinje fibers were perfused with 12.5 microM mexiletine hydrochloride solution and then exposed to hypoxia after 15, 30, 45, or 60 min of perfusion with drug-free solution. Depression of maximal upstroke velocity and shortening of action potential duration persisted during washout, returning to control values by 45 min, although mexiletine was not detectable in the tissue bath after 10 min of washout. Hypoxia initiated at 15 or 30 min of washout failed to produce the anticipated shortening of action potential duration. At 45 and 60 min, action potential duration was shortened by hypoxia. We concluded that mexiletine depression of Vmax and shortening of action potential duration may persist in the absence of drug. Further shortening of action potential duration in response to hypoxia is prevented during this period. The persistence of Vmax depression is prolonged by hypoxia.

Effects of simultaneous administration of mexiletine and quinidine on the electrophysiologic parameters of canine Purkinje fibers.

The electropharmacologic effects of an equimolar combination of mexiletine and quinidine on canine Purkinje action potential parameters were compared with the effects of either drug alone in concentrations ranging from 0.31 to 5.0 X 10(-5) M. Six separate experiments were performed with mexiletine alone, quinidine alone, and the combination. At low concentrations, all three shortened action potential duration (APD) and effective refractory period (ERP) to the same degree; however, the combination depressed Vmax to the same extent as did solutions containing twice the concentration of quinidine alone even though mexiletine alone, in these concentrations, had no effect on the maximum rate of depolarization of phase 0 of the action potential Vmax. In higher concentrations, the combination depressed Vmax and prolonged the ERP to values midway between that achieved by the two drugs separately. Although increasing concentrations of mexiletine alone progressively shortened APD, the combination prolonged this parameter to values similar to those seen with twice the concentration of quinidine alone. The electropharmacologic effects of mexiletine and quinidine in combination are not simply additive and cannot be entirely predicted on the basis of a knowledge of their effects in isolation.

Comparative electropharmacology of mexiletine, lidocaine and quinidine in a canine Purkinje fiber model.

Although said to be lidocaine-like, there is evidence that mexiletine has some properties similar to those of quinidine. To evaluate the relationship between the effects of these three drugs we analyzed their effects on action potentials of Purkinje fibers from canine false tendons. Experiments with each drug were done on single preparations from each of six dogs over the concentration range 0.31 to 10.0 X 10(-5) M. Lidocaine shortened action potential duration and effective refractory period with a log concentration-response relationship. The maximum velocity of phase "0" (Vmax) was unaffected at low concentrations and only slightly depressed at high concentrations. Quinidine also shortened action potential duration and effective refractory period but the effect levelled off at midrange and was reversed at higher concentrations. Quinidine depressed Vmax throughout the concentration range studied. Mexiletine shortened action potential duration with a log concentration response relationship. Effective refractory period was shortened at low concentrations but the effect levelled off and then reversed at higher concentrations of the drug. Mexiletine had no effect on Vmax at the lower end of the concentration curve but did suppress this parameter at higher concentrations. Mexiletine resembled lidocaine in its effect on Vmax and action potential duration whereas the effects of quinidine on these two parameters were distinct. On the other hand, the effects of mexiletine on effective refractory period were more akin to quinidine than lidocaine.