Foster litters prevent hypothalamic-pituitary-adrenal axis sensitization mediated by neonatal maternal separation.

Neonatal maternal separation of rat pups has been shown to produce long-term increases in hypothalamic-pituitary-adrenal (HPA) axis responsiveness, elevated levels of hypothalamic corticotropin releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN), and enhanced anxiety-like behavior. These effects appear to be at least partially mediated by subtle disruptions in the quality of maternal-pup interactions. This hypothesis was tested by providing half the dams with foster litters during the maternal separation paradigm, so that in those litters, only the pups and not the dams were experiencing a period of separation. The separation protocol took place daily from PND2-14 for either 15 min (HMS15, handled) or 180 min (HMS180, maternal separation). During the period of separation dams were either transferred to adjacent cages without any pups present (HMS15, HMS180) or to cages containing an age-matched foster litter (HMS15F, HMS180F). As adults, the HMS180 progeny exhibited the expected increased expression of CRF mRNA in the PVN, stress hyper-responsiveness to airpuff startle and evidence of impaired feedback both in the CORT response, as well as in response to the dexamethasone suppression test. The HMS180F rats, however, appeared to be resistant to these effects of maternal separation as they demonstrated CRF mRNA levels intermediate between HMS15 and HMS180 rats. Their stress responses and feedback regulation of the HPA axis was comparable to that of the HMS15 rats. GR mRNA was elevated in the cortex of HMS180F rats. Overall, these studies support the thesis that the long-term effects of neonatal maternal separation may largely result from alterations in the quality of maternal care rather than from direct effects of the separation per se on the pups.

Negative affect in offspring of depressed mothers is predicted by infant cortisol levels at 6 months and maternal depression during pregnancy, but not postpartum.

This study tests the hypothesis that maternal depression during pregnancy predicts temperament in offspring aged 6 m to 5 y. Previous studies have shown that maternal depression is related to negative affect and that certain temperament factors, such as negative affect and behavioral inhibition, in children predict affective disorders. Here, maternal depression is divided into depression during pregnancy vs. depression postpartum. Maternal depression was determined by the Beck Depression Inventory (BDI) throughout pregnancy and postpartum (prospectively) and by a diagnostic interview (SCID) at 6 months postpartum. The data show that maternal depression during pregnancy, but not postpartum, predicted the ratings of negative affect in the offspring. Importantly, symptoms of depression in the mother (BDI) were used as a control variable in the analyses in order to control for potential bias related to the mother's mood. In addition, cortisol levels in response to a mild stressor at 6 months of age predicted negative affect in infants and toddlers. We conclude that the effects of maternal depression on behavioral problems and vulnerability to mental illness may be mediated by altered temperament and enhanced stress responsiveness.

Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in Long Evans rats and reversal with antidepressant treatment.

RATIONALE: This study was based on the findings of a high comorbidity among anxiety and depression as well as with alcohol abuse. OBJECTIVE: To evaluate first exposure alcohol preference in a rodent model of moderate neonatal maternal separation. METHODS: Rat pups were exposed to either normal animal facility rearing (AFR) or 15 min (HMS15) or 180 min (HMS180) of maternal separation from postnatal days 2-14. The adult (>60 days) male Long Evans progeny was tested for pituitary-adrenal axis responsiveness to airpuff startle, anxiety-like behavior in the elevated plus maze, and alcohol preference using a two-bottle, free-choice test. RESULTS: In response to home cage airpuff startle, HMS180 rats displayed an elevation in the integrated adrenocorticotropic hormone and corticosterone responses. In addition, HMS180 rats spent less time in the open arms and more time in the closed arms in the elevated plus maze. HMS180 rats drank significantly less of a water-sucrose solution and significantly more of an ethanol-sucrose solution than AFR or HMS15 rats. No rearing group differences were observed in total fluid intake. The integrated corticosterone response to airpuff startle was highly correlated with ethanol consumption and there was a negative correlation between percentage of time spent in the open arms of the elevated plus maze and ethanol consumption. Treatment with the selective serotonin reuptake inhibitor paroxetine for 21 days eliminated differences in the elevated plus maze and HPA axis responsiveness, and significantly reduced the amount of ethanol consumed by the HMS180 rats, without affecting these parameters in the HMS15 rats. CONCLUSIONS: These observations suggest that this maternal separation paradigm is a good model to study the effects of early adverse experience on the development of alcohol preference and anxiety.

Stability of motor-unit force thresholds in the decerebrate cat.

To further test the hypothesis that some fixed property of motoneurons determines their recruitment order, we quantified the variation in force threshold (FT) for motoneurons recruited in muscle stretch reflexes in the decerebrate cat. Motor axons supplying the medial gastrocnemius (MG) muscle were penetrated with micropipettes and physiological properties of the motoneuron and its muscle fibers, i.e., the motor unit, were measured. FT, defined as the amount of MG force produced when the isolated motor unit was recruited, was measured from 20 to 93 consecutive stretch trials for 29 motor units. Trials were selected for limited variation in base force and rate of rise of force, which have been shown to covary with FT, and in peak stretch force, which gives some index of motor-pool excitability. Under these restricted conditions, large variation in FT would have been inconsistent with the hypothesis. Analysis of the variation in FT employed the coefficient of variation (CV), because of the tendency for FT variance and mean to increase together. We found that CV was distributed with a median value of 10% and with only 2 of 29 units exceeding 36%. Some of this variation was associated with measurement error and with intertrial fluctuations in base, peak, and the rate of change of muscle force. CV was not significantly correlated with motor-unit axonal conduction velocity, contraction time, or force. In three cases FT was measured simultaneously from two motor units in the same stretch trials. Changes in recruitment order were rarely observed (5 of 121 stretch trials), even when FT ranges for units in a pair overlapped. We suggest that the large variation in recruitment threshold observed in some earlier studies resulted not from wide variation in the recruitment ranking of motoneurons within one muscle, but rather from variation in the relative activity of different pools of motoneurons. Our findings are consistent with the hypothesis that recruitment order is determined by some fixed property of alpha-motoneurons and/or by some unvarying combination of presynaptic inputs that fluctuate in parallel.

The synthesis of N-hydroxy-N'-phenyloctanediamide and its inhibitory effect on proliferation of AXC rat prostate cancer cells.

We have developed a practical synthesis of N-hydroxy-N'-phenyloctanediamide from the methyl ester of suberanilic acid. It provides the product in high yield and purity with a simple purification process. We have found that at 10(-5) M it has a dramatic effect on T/5 AXC/SSh rat prostate cancer cells in vitro. It is a potent inhibitor of cell proliferation and it changes the cell morphology to resemble nonmalignant cells.

Influence of packaging and processing conditions on the decontamination of laboratory biomedical waste by steam sterilization.

The conditions for optimal steam decontamination of polypropylene bags half loaded with laboratory biomedical waste were studied (276 bags were processed). Controls were single-closed bags without water added or incisions made in the top, standing freely in an autoclave set at 121 degrees C. The average time required to reach 121 degrees C at the load center was 46 min for controls. A significant increase in this time occurred following addition of water to bags without incisions (60 min), with double bagging (60 min), or when using vertical containers (82 min). A significant decrease occurred when bags were slashed (37 min) or processed at 123 degrees C (32 min) or 132 degrees C (19 min). Horizontal containers or addition of water to slashed bags had no significant effect.

HIV infection of human gastrointestinal submucosal cells: an in vitro model that mimics Kaposi's sarcoma.

Infection of human gastrointestinal submucosal mesenchymal cells with HIV-1 led to cell populations with abnormal growth properties, increased synthesis of endothelial cell and angioblast markers, and release of angiogenic factors. This system may be the first in vitro model for HIV-induced Kaposi's sarcoma.

Infection of human gastrointestinal cells by HIV-1.

Human immunodeficiency virus (HIV-1) infected and replicated in primary cultures of normal human ileal and colonic epithelial cells. Monocyte-tropic strains (ADA, 24, and 36) were better able to replicate in the gastrointestinal (GI) cells than the T-cell-tropic HIV strain HTLV-IIIB. In some cultures, virus replication persisted through several months. Intestinal epithelium may be an initial target and reservoir for HIV and a vector for virus dissemination and transmission.

Anti-idiotypic antibodies against a monoclonal antibody specific for the trichothecene mycotoxin T-2.

A BALB/c murine monoclonal antibody against the trichothecene mycotoxin T-2 was generated. The antibody, designated HD11, specifically bound T-2 mycotoxin. The binding of HD11 to T-2 conjugated to bovine serum albumin was inhibited by free T-2 toxin but not by the water-soluble heterocyclic guanidines saxitoxin and tetrodotoxin. The T-2 detection limit in an enzyme-linked immunosorbent assay with HD11 was in the nanogram range. The in vitro cytotoxicity of T-2, as measured by the inhibition of radiolabeled leucine uptake of the human epidermoid carcinoma Hep-2 and KB cell lines, was completely reversed by the addition of HD11. Rabbit anti-idiotypic antibodies specific for HD11 were generated and characterized.

Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin.

The sodium channel blocker, tetrodotoxin (TDT), was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Anti-TDT antibodies were detected in serum by ELISA and reached stable levels 4-5 wk after the first immunization. Spleens from immunized mice were fused with NS-1 mouse myeloma cells and approximately 9,329 resultant hybrids were screened by ELISA for reactivity to TDT. Two stable hybrids were isolated, subcloned, and characterized. These hybrids, termed TD13a1 and TD2C5, secreted specific anti-TDT antibodies that recognized TDT but not the related sodium channel blocker, saxitoxin (STX), as determined by competition ELISA. Both antibodies were of the IgG1k subclass with Ka's approaching 10(7) M-1. The inhibitory ability of these antibodies was tested by a competitive displacement assay for [3H]STX on rat brain membranes. Both antibodies strongly inhibited TDT binding to membranes. A nanomole of TD2C5 was able to bind approximately 1.8 nmol of TDT, whereas a comparable amount of TD13a1 bound half as much. Furthermore, TD2C5 was able to protect against TDT-induced reduction of peripheral nerve action potentials in rat tibial nerve when administered in situ. These antibodies thus represent potentially useful reagents for neurobiologic research, detection of toxin contamination and diagnosis of poisoning, and may provide protection against the toxicity of TDT in vivo.

In vitro and in situ inhibition of the sodium channel blocker saxitoxin by monoclonal antibodies.

The sodium channel blocker saxitoxin (STX) was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Anti-STX antibodies were detected in serum by an enzyme-linked immunosorbent assay (ELISA) within a week or two after the first immunization. Spleens from immunized mice were fused with NS-1 myeloma cells and approximately 7000 resultant hybrids were screened by ELISA for reactivity to STX. Two stable hybrids were isolated, subcloned, and characterized. These hybrids, termed S1A5 and S3E.2, secreted specific anti-STX antibodies that did not recognize the closely related toxin tetrodotoxin (TDT), as determined by competition ELISA. The S1A5 monoclonal antibody (mAb) was of the IgMk class and S3E.2 of the IgG1k subclass with affinity constants (Ka values) of approximately 10(6) M-1. The protective ability of these antibodies was tested by a competitive displacement assay for [3H]STX binding on rat brain membranes. Purified S3E.2 strongly displaced [3H]STX binding, whereas S1A5 weakly inhibited [3H]STX binding to membranes. One nanomole of S3E.2 or S1A5 was able to bind 0.03 nmol or 0.005 nmol, respectively, of STX. The S3E.2 mAb offered partial protection against STX-induced reduction of peripheral nerve action potential in rat tibial nerve when administered in situ at concentrations 10- to 30-fold greater than STX. The S1A5 mAb, despite its ability to inhibit STX binding in vitro, was completely ineffectual in situ. These antibodies, particularly S3E.2, thus represent potentially useful reagents for neurobiologic research, detection of toxin contamination, and diagnosis of poisoning, and may provide protection against the toxicity of STX in vivo.

Antiandrogen effects in models of androgen responsive cancer.

The ability of antiandrogens to antagonize androgen effects in androgen responsive tissues is well established. Antiandrogens may diminish in vivo or in vitro proliferation of some androgen responsive cancer cells without causing cessation of multiplication. These model studies are representative of clinical experience in treatment of human prostate cancer with antiandrogen therapy. Recent studies in the AXC/SSh rat prostate cancer model show that these cancer cells elaborate polypeptide growth factors which stimulate their proliferation. If growth factor production by these cells is androgen independent, this may provide an explanation for failure of androgen ablation or antiandrogen treatment to effectively halt prostate cancer cell proliferation.

Differential metabolism of dehydroepiandrosterone sulfate and estrogen conjugates by normal or malignant AXC/SSh rat prostate cells and effects of these steroid conjugates on cancer cell proliferation in vitro.

Normal AXC/SSh rat ventral prostate and clonally derived AXC/SSh rat prostate cancer cells were evaluated for ability to metabolize estrone sulfate (E1S), estrone glucuronide (E1G), or dehydroepiandrosterone sulfate (DHEAS). Both normal and malignant prostate cells converted E1S to estrone. Neither normal nor malignant prostate cells had significant ability to metabolize DHEAS to DHEA, indicating differential specificity of prostate sulfatases(s) for estrogen and androgen sulfates. Both normal and neoplastic prostate cells possess beta-glucuronidase which hydrolyzed E1G to estrone. To assess potential physiologic consequences of these enzymatic activities, we determined the effect of steroid conjugates on in vitro proliferation of selected clonal lines of AXC/SSh rat prostate cancer cells. DHEAS, 10(-6) to 10(-9) M in decade intervals, did not affect in vitro proliferation of AXC/SSh prostate cancer cells; however, 10(-5) M DHEAS decreased in vitro proliferation of these cells. Neither E1S nor E1G, 10(-5) to 10(-9) M in decade intervals, affected in vitro proliferation of AXC/SSh prostate cancer cells. These findings suggest that low residual levels of steroid conjugates, which are not removed by charcoal stripping of serum, do not affect demonstrated in vitro androgen modulation of AXC/SSh rat prostate cancer cell proliferation (Cancer Res. 46, 3775-3781, 1986).

Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated.

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.

Differential modulation of human chorionic gonadotropin production by methotrexate in normal and malignant placental cultures and its increase by dibutyryl cyclic adenosine monophosphate and/or actinomycin D in normal cultures.

The influence of methotrexate (MTX), dibutyryl cyclic AMP, and actinomycin D on production of human chorionic gonadotropin (HCG) in normal first trimester human placental organ cultures was compared. Actinomycin D (10(-8) to 10(-6) M) elevated HCG production by as much as 3.5-fold in normal placenta, and a 2-fold increase in HCG levels was obtained by treatment with dibutyryl cyclic AMP (1 mM) and theophylline (1 mM). The combination of dibutyryl cyclic AMP (1 mM) plus theophylline (1 mM) and actinomycin D (10(-8) M) additively enhanced HCG production by 4.5-fold. In contrast, HCG levels in normal placental organ cultures were unaffected by MTX (10(-8) to 10(-5) M) despite several differing treatment regimens. The JAr line of human choriocarcinoma cells, on the other hand, exhibited an 8-fold increase in HCG levels following MTX exposure (10(-7) M). Incorporation of selected radiolabeled precursors of the de novo and salvage pathways of DNA synthesis was evaluated to assess potential metabolic alterations underlying the differential HCG response of these cultures to MTX. Deoxyuridine incorporation into DNA was decreased similarly in both normal and malignant placenta following MTX exposure. However, deoxycytidine incorporation was inhibited by MTX in normal placental cultures but was elevated by as much as 4-fold in JAr cultures exposed to MTX. Thymidine incorporation into DNA was increased in both groups in the presence of MTX; however, thymidine incorporation was more profoundly stimulated (5-fold) in normal placenta than in JAr cultures (2.5-fold). These data indicate dissimilar utilization of the de novo and salvage pathways of DNA synthesis by these cultures which may explain their differential responsiveness to MTX.

Differential androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro and its antagonism by antiandrogen.

We examined androgen modulation of proliferation of clonally derived AXC/SSh rat prostate cancer cells. C-family cells were maintained on medium without addition of androgens. D-family cells were maintained on medium containing 10(-7) M 5 alpha-dihydrotestosterone and T-family cells were maintained on medium containing 10(-7) M testosterone. Proliferation of all AXC/SSh prostate cancer cell lines during propagation on media containing fetal bovine serum was not altered by changes in media testosterone concentration through the range 10(-6) to 10(-9) M. Similarly, proliferation of C- or D-family cell lines, during propagation on media containing steroid depleted, charcoal stripped fetal bovine serum, was not altered by changes in media testosterone concentration through the range 10(-6) to 10(-9) M. By contrast, proliferation of T-family cell lines during propagation on charcoal stripped fetal bovine serum was modulated by androgens; effects were androgen concentration dependent and maximum at 10(-8) to 10(-7) M. Androgens decreased T5 cell proliferation rate and diminished achievable saturation density, whereas T1 cell proliferation rate was increased by androgens. In contrast, T6 cell proliferation rate was unaffected by androgens; however, saturation density was increased. Effects were antagonized by the antiandrogen RU 23908, Anandron, establishing androgen specificity of testosterone or 5 alpha-dihydrotestosterone mediated changes in proliferation.

Biochemical and morphological characterization of clonal AXC rat prostate cancer cells.

We used three heterogeneous parental cultures of LSC-AXC rat prostate cancer cells: LSC-AXC-C/O, cells maintained on culture medium; LSC-AXC-D/O, cells maintained on culture medium containing 10(-7) M 5 alpha-dihydrotestosterone; and LSC-AXC-T/O, cells maintained on culture medium containing 10(-7) M testosterone, to isolate clonally derived cell lines. Eleven of 15 clonal cell lines were tumorigenic when inoculated into intact male AXC rats. Eight tumorigenic clonal cell lines were selected for further evaluation, and all were found to possess features characteristic of secretory epithelium, as judged by light and electron microscopy. All parental cell lines and the eight selected clonal cell lines contained cytoplasmic and nuclear androgen receptors. Total receptor content was 131 +/- 61 (S.D.), 43 +/- 32, and 274 +/- 96 fmol/100 micrograms of DNA, respectively, for C-, D-, and T-cells. The differences were significant (p less than 0.05). Androgen receptor content of young mature or senescent AXC rat ventral prostate, respectively, is 518 +/- 58 and 266 +/- 40 fmol/100 micrograms of DNA. Since chromosomal analysis established that LSC-AXC prostate cancer cells are hypotriploid, androgen receptor content per cell in C- and T-cells is indicated to be either greater than or equal to that of senescent AXC rat ventral prostate, the tissue in which the original adenocarcinoma arose. Parental and clonal cell lines contained 5 alpha-reductase activity. There were significant differences (p less than 0.05) in both total reductase activity and metabolite distribution. Consequently, the intracellular content of testosterone metabolites was cell line specific. All characterized cell lines contained a higher concentration (p less than 0.05) of APase activity than did young mature or senescent AXC rat ventral prostate. In 6 of 11 cell lines, prostate-secretory APase concentration exceeded (p less than 0.05) that of AXC rat ventral prostate. However, the relative content of secretory APase compared to total APase in carcinoma cells consistently was less (p less than 0.05) than that of AXC rat ventral prostate. These studies document the establishment of clonal AXC rat prostate adenocarcinoma cell lines which have retained important morphological and phenotypic markers characteristic of differentiated prostate epithelium. Since these cells are tumorigenic and represent a spectrum of retained differentiated phenotypic markers, they should be particularly useful for in vivo and in vitro studies of hormonal regulation of prostate cancer cell behavior.

The estimation of hepatic blood flow with indocyanine green: comparison between the continuous infusion and single injection methods.

We compared the estimation of hepatic blood flow obtained using a continuous infusion of indocyanine green with that obtained after a single intravenous injection of indocyanine green in 35 patients with liver disease. There was no significant difference in the values of hepatic blood flow measured by these two methods, and there was a close correlation between the continuous infusion and single injection methods (r = 0.926, p less than 0.001). In a second group of nine patients with cirrhosis, we evaluated the effect of sampling from the right versus left hepatic vein on hepatic blood flow estimation. There was no significant difference between the two estimates and a good correlation was found (r = 0.878, p less than 0.001). Since values of hepatic blood flow measured after a single indocyanine green injection are similar to those measured using the more classical continuous infusion method, the single injection method may be preferable since it requires only 15 min to be performed.