[Electro-convulsive treatment of elderly patients with both behavioral problems and dementia]. / Elektroconvulsieve behandeling van gedragsproblemen bij ouderen met dementie.

Behavioral problems in elderly patients with dementia cannot always be managed with pharmacological or non-pharmacological treatment. We report on three therapy-resistant patients suffering from both behavioral problems and dementia whose condition finally improved after being given electroconvulsive therapy (ECT). Literature data relating to the safety and efficacy of ECT administered to patients with both behavioral problems and dementia are discussed.

Immunogenicity of biopharmaceuticals in laboratory animals.

The potential immunogenicity of new protein therapeutics raises concerns about the possibility of inducing untoward immune reactions in humans. It is generally assumed that all animals will make antibody to human proteins and therefore, there is sentiment among some scientists that this makes the issue of immunogenicity as a safety concern irrelevant. However, recent clinical trials with some proteins have detected the presence of autoantibodies that have resulted in clinical sequelae. These reactions were also observed in preclinical animal studies. In fact, non-human primate and transgenic mouse models can be useful for predicting the relative immunogenicity of human proteins. In addition, the characterization of the immunogenicity of biotechnology molecules provides a practical basis for determining the significance of antibody formation in preclinical safety studies.

The selective estrogen receptor modulator, raloxifene: a segment II/III delivery study in rats.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

Immunogenicity of tissue plasminogen activators in rhesus monkeys: antibody formation and effects on blood level and enzymatic activity.

The immunogenicity of a tissue-type plasminogen activator analog, mt-PA6, consisting of the second kringle and protease domains, was compared to that of the native-sequence protein (nt-PA) in rhesus monkeys. Antibody responses were compared in groups of eight monkeys that were treated by i.v. injection twice, 1 month apart, using doses and regimens chosen to mimic therapy (0.5 mg/kg mt-PA6 bolus, 1.25 mg/kg nt-PA bolus + infusion). An additional group was treated with a 0.5 mg/kg nt-PA bolus. A single positive response was obtained in a monkey treated with 0.5 mg/kg nt-PA after the primary injection. Following the secondary injection, responses were obtained in 1/8, 3/8, and 6/8 monkeys treated with mt-PA6, nt-PA as a bolus, or nt-PA as a bolus + infusion, respectively. Several monkeys were selected to determine whether circulating tPA antibody altered the pharmacokinetics of mt-PA6. Clearance was found to decrease without affecting peak blood levels as antibody concentrations increased from 0.02 to 100 micrograms/ml. In contrast, the peak blood level was reduced by 99% at an antibody concentration of 152 micrograms/ml in a monkey that had been exposed to mt-PA6 in adjuvant 14 months previously. Further, only the serum from this and three other hyperimmunized monkeys inhibited the enzymatic activity of tPA in vitro. It is concluded that mt-PA6 is not more immunogenic than nt-PA in rhesus, and that low levels of antibody are more likely to influence the pharmacokinetic properties of tPA than to inhibit its enzymatic activity. It is unlikely that mt-PA6 would present a serious immunogenic risk in humans.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral and cell-mediated immunity in Sprague-Dawley rats.

There is much discussion about the occurrence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced effects on the human immune system. Extensive studies have been conducted in mice, but those results cannot explain some of the epidemiological data obtained in exposed humans. Therefore, studies in other laboratory animal species are needed. The aim of these experiments was to examine effects of TCDD on cell- and humoral-mediated immunity in male Sprague-Dawley (SD) rats. A delayed-type hypersensitivity (DTH) assay was used to examine cell-mediated immunity. A time-course study demonstrated that TCDD treatment on day -5 relative to immunization (day 0) produced the greatest effect on cell-mediated immunity. In a dose-response experiment, rats were treated with 1, 3, 10, 20, 30, 40 and 90 micrograms TCDD/kg The effect of TCDD on cell-mediated immunity displayed an inverted U-shaped dose-response curve, in that low doses enhanced and high doses suppressed this immune function. This is the first study to demonstrate an U-shaped dose-response curve of TCDD on the immune system. Primary antibody response to sheep red blood cells (SRBC) was used as endpoint to study the effect of TCDD on humoral immunity. Serum anti-SRBC IgM and IgG levels were measured using an enzyme-linked immunosorbent assay (ELISA). In the dose range examined (10, 20 and 40 micrograms TCDD/kg), serum IgM levels were not affected by TCDD compared to controls at 7 and 14 days after immunization. In contrast, serum IgG levels were dose-dependently elevated both 7 and 14 days after immunization, with a maximum increase of 59% over controls.

Immunogenicity of biosynthetic human LysPro insulin compared to native-sequence human and purified porcine insulins in rhesus monkeys immunized over a 6-week period.

Development of insulin antibodies in rhesus monkeys was investigated after immunization with 3 forms of insulin in Freund's adjuvant. Insulins examined included: 1. biosynthetic LysPro insulin (LY275585), a new human insulin analog, 2. biosynthetic native-sequence human insulin, and 3. purified porcine insulin. Male monkeys, 4/insulin type, were immunized weekly over a 6-week period with increasing doses of insulin, ranging from 10 to 100 micrograms/monkey. An ELISA assay was used to measure IgG insulin antibodies in sera collected prior to immunization and 5, 10, and 16 days after final immunization. One monkey had detectable pretreatment levels of antibody. This monkey, which had been assigned to the LysPro insulin treatment group, responded to immunization with a peak antibody level of 20 micrograms/ml. IgG insulin antibody responses were not detected in any of the other monkeys. A passive cutaneous anaphylaxis (PCA) assay was used to measure IgE insulin antibodies in sera collected prior to immunization and 10 days after final immunization. No IgE antibodies were detected in any of the monkeys pre- or post-immunization. Considering that 1. an immunological adjuvant was used, 2. eleven of twelve monkeys failed to develop an antibody response, and 3. the IgG insulin antibody level observed in the single responding monkey was low, it was concluded that these insulins have an extremely weak immunogenic potential in rhesus monkeys. It is suggested that immunization of non-human primates with new therapeutic proteins in adjuvant may be a useful primary screen to determine their immunogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in production of interleukin-7 (IL-7) by murine long-term bone marrow cultures (LTBMC).

Age-related decreases in humoral immune function have been well documented. In aged mice, these functional deficits may be due, in part, to decreased numbers of precursor B cells. Interleukin-7 (IL-7) plays a key role in B cell development by stimulating proliferation of progenitor and pre-B cells. In the current study, proliferation of the murine IL-7-dependent pre-B cell line SCID/FC-7 (SCID) was used to assess IL-7 activity in long-term bone marrow culture-conditioned medium (LTBMC-CM) from both young (4-8-week-old) and older (16-40-week-old) mice. Time to reach peak production and peak IL-7 levels were similar in both groups and was optimal between weeks 2 and 6 of culture. IL-7 activity in LTBMC-CM from older mice fell rapidly to negligible levels after 8 weeks in culture. These findings are consistent with age-related changes in stem cell production and B lymphopoiesis in LTBMC reported in other studies.

Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys.

The relative concentrations of antibodies produced in monkeys against three forms of human growth hormone (hGH) were determined using an antigen-specific avidin/biotin ELISA assay. Monkeys were treated in two separate 90-day studies with recombinant methionyl-hGH (met-hGH) and pituitary-derived hGH (pit-hGH) (Study 1) and recombinant natural sequence hGH (Study 2). The lowest dose was equal to the expected therapeutic dose of 0.1 IU/kg. Sixty-nine percent of monkeys treated with pit-hGH and 81% of those treated with met-hGH developed detectable anti-hGH responses. The magnitudes of the responses exhibited wide animal to animal variability, were not markedly related to dose or sex, and were lower than levels obtained in monkeys immunized with hGH in Freund's adjuvant. In contrast, the incidence of antibody responses in monkeys treated with natural sequence hGH was lower (23% in one experiment and 5% in a replicate experiment) and took longer to develop. Antibody concentrations were lower, on average, than in those animals treated with met- or pit-hGH. These results are in accord with those observed clinically, thus supporting the use of the monkey model to predict the relative immunogenicity of some proteins in humans.

Environmentally related disorders of the hematologic and immune systems.

From observations in rodents and, to a lesser extent, in humans inadvertently or occupationally exposed, it appears that a number of xenobiotics adversely affect immune homeostatic systems, either through acting as a hapten and resulting in hypersensitivity reactions or through altering hematopoietic or immune functions. At present, however, there is no evidence that the immune or hematopoietic systems of the general population have been compromised by xenobiotics via environmental exposure. Nonetheless, these examples and our current knowledge about the pathogenesis of disease support the possibility that chemical-induced damage to the immune system may be associated with potential pathological conditions, some of which may become detectable only after a long latency. Likewise, exposure to immunotoxic xenobiotics might represent additional risk to individuals with already fragile immune systems (e.g., in malnutrition, infancy, old age). However, it is important to be cautious when attempting to extrapolate meaningful conclusions from experimental data or isolated epidemiologic studies to risk assessment for low-level human exposure.

Bone marrow stromal cell bioactivation and detoxification of the benzene metabolite hydroquinone: comparison of macrophages and fibroblastoid cells.

Bone marrow stroma consists predominately of two cell types, macrophages and fibroblastoid stromal cells, which regulate the growth and differentiation of myelopoietic cells via the production of growth factors. We have previously shown that macrophages are more sensitive than fibroblastoid stromal cells (LTF cells) to the toxic effects of the benzene metabolite hydroquinone. In this study, the role of selective bioactivation and/or deactivation in the macrophage-selective effects of hydroquinone was examined. LTF and macrophage cultures were incubated with 10 microM [14C]hydroquinone to examine differential bioactivation. After 24 hr, the amount of 14C covalently bound to acid-insoluble macromolecules was determined. Macrophages had 16-fold higher levels of macromolecule-associated 14C than did LTF cells. Additional experiments revealed that hydroquinone bioactivation to covalent-binding species was hydrogen peroxide dependent in macrophage homogenates. Covalent binding in companion LTF homogenates was minimal, even in the presence of excess hydrogen peroxide. These data suggest that a peroxidative event was responsible for bioactivation in macrophages and, in agreement with this, macrophages contained detectable peroxidase activity whereas LTF cells did not. Bioactivation of [14C]hydroquinone to protein-binding species by peroxidase was confirmed utilizing purified human myeloperoxidase in the presence of hydrogen peroxide and ovalbumin as a protein source. High performance liquid chromatographic analysis of incubations containing purified myeloperoxidase, hydroquinone, and hydrogen peroxide showed that greater than 90% of hydroquinone was removed and could be detected stoichometrically as 1,4-benzoquinone. 1,4-Benzoquinone was confirmed as a reactive metabolite formed from hydroquinone in macrophage incubations using excess GSH and trapping the reactive quinone as its GSH conjugate, which was measured by high performance liquid chromatography with electrochemical detection. The activity of DT-diaphorase, a quinone reductase that has been invoked as a protective mechanism in quinone-induced toxicity, was 4-fold higher in LTF cells than macrophages. These data suggest that the macrophage-selective toxicity of hydroquinone results from higher levels of peroxidase-mediated bioactivation and/or lower levels of DT-diaphorase-mediated detoxification.

Activation and deactivation of quinones catalyzed by DT-diaphorase. Evidence for bioreductive activation of diaziquone (AZQ) in human tumor cells and detoxification of benzene metabolites in bone marrow stroma.

Bioactivation of diaziquone (AZQ) in HT-29 human colon carcinoma cells and detoxification of benzene metabolites in bone marrow stromal cells were used as examples of the potential role of DT-diaphorase in both activation and deactivation processes. HT-29 cell cytosol contained high levels of DT-diaphorase activity and removed AZQ in the presence of either NADH or NADPH. Prior boiling of cytosol, omission of NADH or NADPH or inclusion of dicoumarol, an inhibitor of DT-diaphorase, inhibited removal of AZQ. AZQ-induced cytotoxicity in HT-29 cells was also inhibited by dicoumarol. Chemical reduction of AZQ in a cell free system enhanced formation of a GSH conjugate of AZQ. Two of the major cell types in bone marrow stroma are macrophages and fibroblastoid stromal cells. A fibroblastoid cell line derived from stromal cells contained approximately fourfold higher levels of DT-diaphorase than macrophages. Inclusion of dicoumarol in incubations containing 14C-hydroquinone and the respective stromal cell type, significantly increased covalent binding of radiolabel to macromolecules in stromal fibroblasts but not in macrophages.

Bone marrow stromal cell regulation of B-lymphopoiesis. II. Mechanisms of hydroquinone inhibition of pre-B cell maturation.

Previous investigations from our laboratory have shown that the benzene metabolite, hydroquinone (HQ), inhibits B cell production by preventing the maturation of pre-B cells. Data presented in this paper demonstrate that HQ interrupts B-lymphopoiesis indirectly by inhibiting the production of interleukin-4 (IL-4) by fibroblastic stromal cells. HQ exposure of fibroblastic stromal cells (SCL-173 and SCL-160) did not affect IL-4 production by these cell lines at any dose tested. Addition of untreated bone marrow-derived macrophages to HQ-treated bone marrow stromal cells (heterogeneous population of fibroblastic cells and macrophages) reversed inhibition of IL-4 production. However, addition of HQ-treated macrophages was without effect. Our studies suggest that HQ inhibits macrophage production of IL-1, a potent inducer of IL-4 production by bone marrow fibroblastic stromal cells. Interruption of IL-1 release from macrophages mediates the observed inhibition of B lineage cell maturation.

Macrophage regulation of myelopoiesis is altered by exposure to the benzene metabolite hydroquinone.

Hydroquinone, a myelotoxic metabolite of benzene, decreases the ability of murine bone marrow stromal cells to support myelopoiesis in vitro. Bone marrow stroma consists of macrophages and fibroblastoid stromal cells that participate coordinately in regulating myelopoiesis. The goal of this study was to determine if macrophage or fibroblastoid cell function is more sensitive to the myelotoxic actions of hydroquinone. To address this question, we developed purified populations of macrophages and fibroblastoid stromal cells and treated each population with hydroquinone. These cells were reconstituted together with nontreated cells of the opposite type and assayed for their ability to support the formation of granulocyte and macrophage colonies in an agar overlay. Reconstituted cultures containing hydroquinone-treated macrophages supported fewer colonies than did corresponding cultures containing untreated macrophages. Reconstituted cultures containing hydroquinone-treated fibroblastoid stromal cells were not affected. Moreover, hydroquinone reduced detectable interleukin-1 activity in purified macrophage cultures stimulated with lipopolysaccharide. These results indicate that hydroquinone selectively interferes with macrophage function possibly, in part, via alteration of macrophage interleukin-1 secretion.

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation.

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.

Chlorphentermine suppresses the phosphatidylinositol pathway in concanavalin A-activated mouse splenic lymphocytes.

We have previously demonstrated that the chlorphentermine (CP)1-induced impairment in lymphocyte blastogenesis involves drug-induced inhibition of an event which occurs very early during lymphocyte activation. An early event, which is associated with mitogen-induced lymphocyte activation, involves the hydrolysis of phosphatidylinositol by phospholipase C to yield inositol phosphates and diacylglycerol as products. Inositol phosphates and diacylglycerol then function as mediators of a trans-membrane signal for the continuation of the cellular response. It was the purpose of the present study to determine the effects of CP on this phosphatidylinositol pathway. We demonstrated that formation of inositol phosphates in lymphocytes increases progressively above control over a 2 hour period following concanavalin A (Con A)-stimulation. In contrast, lymphocytes pre-incubated with 10(-5)M CP for 60 min, then stimulated with Con A for 2 hours in the presence of 10(-5)M CP, exhibit a significantly depressed inositol phosphate formation. In addition, CP also inhibited the activity of phospholipase C (IC50 = 0.58 mM), the enzyme responsible for the formation of inositol phosphates during lymphocyte activation. Further, lymphocytes activated in a manner that bypasses the phosphatidylinositol pathway are not inhibited by 10(-7)M or 10(-9)M CP as are cells activated with Con A. These results suggest that the suppression of the phosphatidylinositol pathway may be involved in the inhibition by CP of lymphocyte blastogenesis induced by Con A.

Hydroquinone inhibits bone marrow pre-B cell maturation in vitro.

Environmental exposure to benzene results in both myelotoxicity and immunotoxicity. Although benzene-induced immunotoxicity has been well documented, no studies to date have addressed the possibility that benzene toxicity is due in part to altered differentiation of marrow lymphoid cells. We investigated the effect of acute exposure to the benzene metabolite, hydroquinone, on murine bone marrow B-lymphopoiesis. Bone marrow cell suspensions from B6C3F1 (C57BL/6J x C3H/HeJ) mice were depleted of mature surface IgM+ (sIgM+) B cells and cultured for 0, 24, 48, or 72 hr and production of newly formed B cells was assayed both by sIgM expression and colony formation in soft agar cultures. One hr exposure of bone marrow cells to hydroquinone before culture reduced the number of sIgM+ cells generated in liquid cultures. Small pre-B cells (cytoplasmic mu heavy chain+, sIgM-) were numerically elevated as compared with control cultures. Hydroquinone exposure also decreased the number of adherent cells found in cultures of bone marrow cells. These results suggest that short-term exposure to hydroquinone, an oxidative metabolite of benzene, may in some way block the final maturation stages of B cell differentiation. This apparent differentiation block resulted in reduced numbers of B cells generated in culture and a corresponding accumulation of pre-B cells. Reduction of adherent cells in treated cultures may also suggest that toxicity to regulatory cells for the B lineage may be in part responsible for this aspect of hydroquinone myelotoxicity.

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin.

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.

The effects of plastic bag extracts prepared in either mouse serum or phosphate-buffered saline with ethanol on mouse splenic cells.

Chemicals leached from plastic blood bags are potentially immunogenic when introduced parenterally into humans. The experiments reported here represent an attempt to sensitize B6C3F1 mice to parenteral injections of solutions used to extract potentially immunogenic material from plastic blood bags. Mice were given intraperitoneal injections: blood bag extracts prepared from phosphate-buffered saline and ethanol; mouse red cells stored in extract solution; or blood bag extracts prepared from mouse serum. None elicited a significant in vitro immunologic reaction as determined by 3H-thymidine or 3H-leucine uptake into spleen cells. In contrast, intraperitoneal injections in mice of large amounts (0.5 ml) of the phosphate-buffered saline and ethanol extracts decreased spleen cellularity to 59 percent of the control value and reduced spleen cell blastogenic activity in vitro. When solutions of either mouse serum extract or mouse cells stored in extract were given intraperitoneally to mice, no spleen toxicity or increased blastogenic activity was observed. In addition, the extracts obtained from plastic blood bags by using either allogenic serum or buffered saline and ethanol did not elicit an immunologic response when they were administered parenterally to mice.