Assay in the mouse for delayed-type hypersensitivity to murine leukemia virus.

Delayed-type hypersensitivity (DTH), assayed by footpad swelling, was induced in 6- to 8-week-old BALB/cCr mice immunized with formalin-inactivated, sucrose-banded murine type-C viruses. The DTH response was inducible with as little as 11.25 mug sensitizing antigen, was greatest after sc sensitization as compared to im and ip sensitization, and was optimally elicited with a 7-day challenge. A statistical evaluation of the DTH assay revealed that the test was consistently reproducible and limited only by biologic variability of the mouse and the standardization of the antigen preparation. The DTH response was specific for type-C virus subtypes because it could distinguish the Rauscher strain of murine leukemia virus from AKR leukemia virus when the challenge antigen was extracted with Tween 80-ether. Immunized mice that gave DTH responses were resistant to challenge with exogenous, live murine leukemia viruses.

Effect of three retinoids on tracheal carcinogenesis with N-methyl-N-nitrosourea in hamsters.

Male Syrian golden hamsters received 12 weekly intratracheal exposures to 0.5% N-methyl-N-nitrosourea with a special catheter. Following exposures, animals were randomized into 4 groups of 63 hamsters and placed on diets of lab meal or meal with 120 mg 13-cis-retinoid acid (CRA)/kg, 327 mg ethyl retinamide (ER)/kg, or 343 mg N-(2-hydroxyethyl)retinamide (HR)/kg for 6 months at which time all hamsters were killed. The observed incidences of tracheal epithelial neoplasms (No. of animals with tumors/total No. of animals) were 10/63 (lab meal), 22/61 (CRA), 24/63 (ER), and 17/62 (HR). The incidence of carcinomas (No. of animals with tumors/total No. of animals) were 4/63 (lab meal), 12/61 (CRA), 12/63 (ER), and 11/62 (HR). The weight loss and mortality relative to those in the group fed the lab meal were significantly in the group fed HR but not in the other retinoid-treated groups.

Natural occurrence of tumors in mouse strains with different xenotropic and ecotropic endogenous viruses.

Natural tumor incidence and type C virus expression in HIH Swiss and BALB/c mice were investigated. The BALB/c mice showed a moderate incidence of lymphoreticular tumors containing infectious ecotropic virus. A second class of lymphoreticular tumors occurred with approximately the same incidence in both strains; though infectious virus could not be isolated from it, it contained the antigens of a common xenotropic virus. These xenotropic viral antigens were found in all NIH Swiss and BALB/c tumors examined and in normal hematopoietic tissues as well. The relationship between different classes of endogenous viruses and tumor development was discussed.

Development of a lymphocyte transformation microassay for murine leukemia virus.

A lymphocyte transformation microassay (LTA) was developed from spleen harvests of 6- to 8-week-old BABL/cCr mice. The optimal culture conditions for the microassay were established by measurement of lymphoblastogenesis in response to phytohemagglutin (PHA) and pokeweek mitogen. Immunization, as measured by the LTA, of adult BALB/cCr mice with formalin-inactivated, sucrose-banded, murine type-C viruses was achieved with a three-dose regimen of 200, 100, and 100 mug during 3 successive weeks (Freund's complete adjuvant was used with the first dose). The ip route of immunization induced the best responses in lymphocytes harvested 18 days after the last immunogen was given. The LTA was consistently reproducible, limited only by biological variability of the mouse and the standardization of the antigen preparation. In mice immunized with Rauscher murine leukemia virus (R-MuLV) or AKR MuLV vaccine, the LTA was specific for the C-type virus and could be used to distinguish viral subtypes, because R-MuLV elicited responses significantly different from a B-tropic BALB/c leukemia virus. This specificity was evident when the stimulating antigen was presented as UV-inactivated, sucrose-banded virus or as freeze-thaw extracts of cell infected with MuLV.

Sensitivity to two-stage carcinogenesis of SENCAR mouse skin grafted to nude mice.

SENCAR mice are extremely susceptible to two-stage skin carcinogenesis, while BALB/c mice are relatively resistant. Skin grafts to BALB/c nude mice were performed with full-thickness skin from SENCAR and BALB/c donors, and tumor formation was monitored in grafted skin, surrounding host skin, and intact SENCAR, BALB/c and nude mice. Initiation was accomplished by exposure to 20 micrograms dimethylbenz(a)anthracene and promotion by repeated exposure to 12-O-tetradecanoylphorbol-13-acetate. SENCAR skin retained a high sensitivity to carcinogenesis when grafted to nude hosts, whereas BALB/c skin remained resistant. The donor type did not influence the tumor yield in surrounding nude host skin. The rate of tumor regression was not altered in SENCAR skin grafts on nude mice relative to intact SENCAR skin. These results indicate that the unusual sensitivity of SENCAR epidermis to chemical carcinogenesis is not due to altered systemic factors but is a property of the skin itself.

An approach to C-type virus immunoprevention of spontaneously occurring tumors in laboratory mice.

A review of our current progress in C-type virus vaccine research is presented. This includes the findings of C-type virus or its antigen expressions in every naturally occurring tumor of two strains of "low-incidence" laboratory mice, the BALB/cCr mouse and the NIH Swiss mouse. Vaccine preparation methods are described including the inactivation of C-type virus infectivity with optimal maintenance of the antigen titers of at least two of the polypeptides of the C-type virus, gp69/71 and p30. The cell-mediated immune response of the mouse to C-type virus vaccines, as measured by a footpad assay for delayed-type hypersensitivity and an in vitro lymphocyte transformation assay, is described. Studies with two murine C-type viruses (Rauscher leukemia and Gross leukemia) a simian C-type virus, and an avian C-type virus (avian myeloblastosis virus) showed that the cell-mediated immune response of the animal includes type-specific, group-specific, and interspecies-specific reactivity. The mouse gave a cell-mediated immune response to at least one of the polypeptides of the C-type virus, the gp69/71, whether this polypeptide was presented to the immune system of the mouse as whole virus, Tween-ether-treated virus, or a purified polypeptide. One measure of the effectiveness of the C-type virus vaccines was provided by immunization of the mouse with Rauscher leukemia virus preparation that induced resistance to challenge with both live Rauscher leukemia virus and a naturally occurring BALB/c leukemia virus. Evidence is presented that the C-type virus can act as an effective transplantation antigen in syngeneic tumor cell lines resulting in the immunogenicity and loss of tumorigenicity of these cell lines. An approach to the viral immunoprevention of spontaneously occurring tumors is discussed.

Opiates as potential cofactors in progression of HIV-1 infections to AIDS.

Because of the widely documented association of AIDS with opiate abuse, there is considerable interest in knowing whether opiates alter progression of HIV-1 infections to AIDS. The main reason for this interest is that opiates and opiate-abuse have been shown to have broad influence on immune processes as well as in vitro expressions of HIV-1. This article reviews literature defining the connection between opiate use and AIDS. Basic understanding of the effects of opiates on immune process and HIV-1 infection, especially as derived from study of a monkey model of AIDS, are discussed as well as epidemiological data regarding the connection between chronic injected drug abuse and AIDS, in the context of current knowledge about the HIV-1 infectious process and AIDS pathogenesis. Theoretically, there is ample reason to suspect that opiates are involved in progression of HIV-1 infections to AIDS. To date, however, epidemiological approaches have been unable to link decline in CD4 T-cell counts, as a marker of AIDS progression, with opiate use--although other indices of AIDS progression have yet to be thoroughly evaluated in this regard. Also, the impact of opiate use and abuse on opportunistic infections occurring prior to or concurrent with HIV-1 infection has not been closely scrutinized. Interestingly, despite considerable evidence delineating the potential of opiates to exacerbate HIV-1 infections, there is suggestive evidence from both clinical observations and basic studies that homeostatically balancing conditions of chronic, consistent opiate exposure have the potential to protect the host from progression of HIV-1 infections--a situation that may well differ from when opiate-naive subjects first experience exposure to opiates and when opiate dependency is not maintained in a consistent fashion. Taken together, therefore, information from basic studies, including most particularly studies with monkeys, and epidemiological studies, indicates that effects of opiates on progression to AIDS may be conditionally variable. There are many aspects of the drug abuse culture that have potentially offsetting consequences in terms of their potential to up- or down-regulate both HIV-1 expression and host protective responses thereto that could be relevant in this regard. In conclusion, many ambiguities are yet to be considered, and basic and epidemiological studies to be pursued before the opiate-AIDS connection is fully understood.

Retinoic acid promotion of papilloma formation in mouse skin.

Retinoic acid is a weak promotor of skin tumorigenesis in Charles River CD-1 mice. Multiple papillomas were seen in 17% of the mice treated 3 times weekly with 5.1 micrograms retinoic acid for 20 weeks after initiation by a single treatment with 50 micrograms 7,12-dimethylbenz[alpha]anthracene (DMBA). These results suggest the necessity of a more thorough evaluation of retinoids as tumor promoters before their serious consideration as anti-cancer agents in man.

Effects of norepinephrine, HIV type 1 infection, and leukocyte interactions with endothelial cells on the expression of matrix metalloproteinases.

The expression of matrix metalloproteinases (MMPs) associated with AIDS-related cardiomypathies and cocaine abuse was examined in an in vitro coculture model. Human peripheral blood mononuclear cells (PBMCs), HIV infected or uninfected, were placed in coculture with primary human cardiac microvascular endothelial cells (HMVEC-C) in the presence or absence of the cocaine-inducible catecholamine norepinephrine (NE). Culture supernatants were assayed for MMP-1, -2, -3, -7, -9, and -13, and for tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2, by enzyme-linked immunosorbent assay. Low levels of constitutively expressed MMP-1 and -2 were detected in individual cultures of HMVEC-C and PBMCs. NE did not induce MMP or TIMP expression by HMVEC-C and caused modest increases (3- to 4-fold) in MMP-1 and -2 by uninfected PBMCs. Increased levels of NE-induced MMP-1 (5-fold) and MMP -2 (15-fold) were detected in cocultures of HMVEC-C and uninfected PBMCs. HIV infection enhanced MMP-1 (46-fold) and MMP-2 (48-fold) and active MMP-7 (33-fold) and MMP-9 (50-fold) by PBMCs. Coculture of HIV-infected PBMCs with HMVEC-C increased MMP-1 (110-fold) and MMP-2 (307-fold) but not active MMP-7 and -9. The combination of NE, HIV infection, and coculture increased MMP-1 (126-fold) and MMP-2 (467-fold), and active MMP-7 (65-fold) and MMP-9 (75-fold). MMP-3 or-13 was not detected in any of the treatment groups and TIMP-1 and -2 appeared inversely proportional to the observed levels of MMPs. These results suggest that HIV infection, NE, and leukocyte endothelial interactions demonstrate separate and overlapping cooperative effects on the regulation of expression of TIMPs and MMPs associated with AIDS-related cardiomyopathies.

Binding of naloxone to human T lymphocytes.

Purified T lymphocytes have a specific binding site for naloxone, the opiate antagonist. The KD for the site was 50.6 +/- 2.4 nM, while the Hill coefficient (n) was 1.67 +/- 0.16, indicating a degree of positive cooperativity of ligand binding. The bound naloxone was partially displaceable by various opiate agonists including morphine (56%), beta-endorphin (61%), met5- and leu5-enkephalin (40% each), [D-ala2, D-leu5]-enkephalin (78%) and [D-ala2, D-leu5]-enkephalinamide (66%). Virtually all of the binding capacity was recovered in the particulate membrane fraction after sonic lysis of the cells. There was great interindividual variability in Bmax between samples, suggesting a possible mechanistic basis for the variability in drug action seen between different individuals.

Norepinephrine enhances adhesion of HIV-1-infected leukocytes to cardiac microvascular endothelial cells.

Recent reports have indicated that norepinephrine (NE) enhances HIV replication in infected monocytes and promotes increased expression of select matrix metalloproteinases associated with dilated cardiomyopathy (DCM) in vitro in co-cultures of HIV-infected leukocytes and human cardiac microvascular endothelial cells (HMVEC-C). The influence of NE on HIV infection and leukocyte-endothelial interactions suggests a pathogenic role in AIDS-related cardiovascular disease. This study examined the effects of norepinephrine (NE) and HIV-1 infection on leukocyte adhesion to HMVEC-C. Both flow and static conditions were examined and the expression of selected adhesion molecules and cytokines were monitored in parallel. NE pretreatment resulted in a detectable, dose-dependent increase of leukocyte-endothelial adhesion (LEA) with both HIV-1-infected and -uninfected peripheral blood mononuclear cells (PBMCs) relative to media controls after 48 hr in co-culture with HMVEC-C in vitro. However, the combination of NE plus HIV infection resulted in a significant (P < 0.0001) 18-fold increase in LEA over uninfected media controls. Increased levels in both cell-associated and -soluble ICAM-1 and E-Selectin but not VCAM-1 correlated with increased LEA and with HIV-1 infection or NE pretreatment. Blocking antibodies specific for ICAM-1 or E-Selectin inhibited HIV-NE-induced LEA. These data suggest a model in which NE primes HIV-1-infected leukocytes for enhanced adhesion and localization in HMVEC-C where they can initiate and participate in vascular injury associated with AIDS-related cardiomyopathy.

The morphine-binding site on human activated T-cells is not related to the mu opioid receptor.

Mitogen activation of human T-lymphocytes induces a morphine-binding site. Morphine binding is displaceable by beta-endorphin (1--31) and (--)-naloxone but not DAMGO. This site is not stereoselective for (--)-morphine. T-lymphocytes, expressing this binding site, were assayed by reverse-transcription polymerase chain reaction (RT-PCR) for expression of hMOR-1 mRNA. Several primer sets were used and each assay compared with cells known to express human or mouse MOR-1 mRNA. Neither hMOR-1 nor any homologous receptor was detected in human T-lymphocytes. Therefore, the morphine-binding site on mitogen-activated T-lymphocytes is unlikely to be closely related to hMOR-1.

Atrophy induced in the tubules of the testes of Syrian hamsters by two retinoids.

This paper describes toxic lesions induced in the hamster testes by 3 different retinoids. Groups of 13 Syrian Golden hamsters were fed diets containing 120 mg/kg 13-cis-retinoic acid (CRA), 327 mg/kg ethyl retinamide (ER), or 343 mg/kg 2-hydroxyethyl retinamide (HER) for 6 months. The germinal epithelium of the testicular tubules was completely atrophic in the groups fed ER and HER. Mean testicular weights were 0.8 g and 0.7 g respectively as compared to 3.3 g in the control group. No alterations in testicular weights or morphological characteristics, at the light microscopic level, were found in the group fed CRA. At the ultrastructural level, however, asymmetrical head caps and deformed acrosomes were observed in the spermatids.

Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts.

To refine previous studies of chromosome damage (CD) and sister-chromatid exchanges (SCE) in heroin addicts, we applied new methods developed in our laboratory to enhance detection of the cytogenetic effects of low-level radiation exposure in hospital workers. For CD analysis, we applied our thymidine-fluorodeoxyuridine-caffeine (TFC) enhancement procedure in which cells at setup receive 1 x 10(-7) M fluorodeoxyuridine to inhibit thymidylate synthetase and 4 X 10(-5) M thymidine to satisfy the induced requirement, and then in G2 receive 2.2 mM caffeine to modulate DNA repair. For SCE enhancement, caffeine treatment was initiated in G1 at 19 h before harvest. Using both standard and enhanced procedures for CD and SCE analysis, blood samples were evaluated from 20 street heroin addicts and 22 controls. Standard 2-day CD and 3-day SCE assays showed small, insignificant genotoxic increases in addicts while the enhanced CD and SCE assays showed highly significant increases. Most CD events were in the form of chromatid and chromosome breaks. There were no rings and only a few dicentrics were observed in the TFC-enhanced cultures. Although quadriradials are rare, 10 were found in addict TFC-cultures and 3 in control TFC-cultures. With the standard CD assay, the mean number of chromosome breaks per 100 cells was 0.727 for controls and 1.056 for addicts (not significant). With the TFC-enhanced assay, the same measure showed 1.483 chromosome breaks for controls and 5.143 for addicts (highly significant, ANOVA: p less than 0.0001). A highly significant difference was also observed for chromatid-type damage with the TFC-enhanced assay (chromatid breaks per 100 cells: 16.793 for controls; 48.191 for addicts). The SCE data also showed significant differences with the enhanced assay. Scoring 25 cells/condition, standard SCE cultures showed 10.892 SCE/cell for controls and 11.732 SCE/cell for addicts (not significant). With CAF enhancement there were 13.08 SCE/cell for controls and 17.05 SCE/cell for addicts (ANOVA: p less than 0.008). These findings indicate that detection of CD and SCE effects can be significantly enhanced by the use of these new procedures. The finding of greatly increased chromatid damage in the addicts with the TFC procedure suggests that at least part of the CD detected occurred in vitro and is not a product of prior in vivo damage. Therefore exposure to this drug and perhaps other environmental agents may not only leave a residue of DNA or chromosome damage but may also induce a sensitivity to further genotoxic damage that is revealed by using the enhanced procedures.

Effects of morphine on T-cell recirculation in rhesus monkeys.

A 2-yr study on effects of morphine on lymphocyte circulation in rhesus monkeys (Macaca mulatta) showed that, over time, a well-maintained morphine-dependency caused biphasic depressive effects on circulating lymphocyte levels. Depression of T cell circulation by opiates actually was a relative effect. Morphine exposure basically stabilized T cell circulation in the context of concurrent increases in controls. Biphasic effects of morphine were attributable to distinctions in circulation kinetics of CD4+/CD62L (+ & -) T cells. That is, levels of CD4+/CD62L+ T cells were selectively depressed by opiates through the first 32wk after initiation of drug, and levels of CD4+/CD62L- T cells were selectively depressed thereafter. Regression analyses also showed that morphine stabilized lymphocyte recirculation. Circulating levels of resting and activated-memory types of T cells were positively correlated in opiate-exposed monkeys during the first 32wk after opiate exposure--an effect not seen with control monkeys. Considerations of changes in the types of experimental stressors extant during the study suggested that temporally differential effects of opiates on T cell recirculation were connected with changes in the stress environment and the ability of morphine to modulate these changes. Thus, morphine, and by inference the endogenous opioid system, are involved in homeostasis of lymphocyte recirculation, probably through effects on central mediation of the stress axis.

Randomised clinical trial: dried plums (prunes) vs. psyllium for constipation.

BACKGROUND: Treatment of chronic constipation remains challenging with 50% of patients dissatisfied with current therapy. There is an unmet need for natural and safe alternatives. Dried plums (prunes) have been used traditionally for constipation but their efficacy is not known. Aim To assess and compare the effects of dried plums and psyllium in patients with chronic constipation. METHODS: Subjects were enrolled in an 8-week, single-blind, randomised cross-over study. Subjects received either dried plums (50 g b.d., fibre=6 gm/day) or psyllium (11 g b.d., fibre=6 gm/day) for 3 weeks each, in a crossover trial with a 1-week washout period. Subjects maintained a daily symptom and stool diary. Assessments included number of complete spontaneous bowel movements per week, global relief of constipation, stool consistency, straining, tolerability and taste. RESULTS: Forty constipated subjects (m/f=3/37, mean age=38 years) participated. The number of complete spontaneous bowel movements per week (primary outcome measure) and stool consistency scores improved significantly (P<0.05) with dried plums when compared to psyllium. Straining and global constipation symptoms did not differ significantly between treatments (P=N.S.). Dried plums and psyllium were rated as equally palatable and both were safe and well tolerated. CONCLUSION: Dried plums are safe, palatable and more effective than psyllium for the treatment of mild to moderate constipation, and should be considered as a first line therapy.