Leukemia virus-induced immunosuppression: scanning electron microscopy of infected spleen cells.

Spleen cells from mice infected with Friend leukemia virus were examined by scanning electron microscopy. Whereas splenocytes from normal noninfected animals showed the expected morphologic classes of lymphocytes, including those with smooth surfaces and with numerous villous projections, an alteration of cell type was evident within a few days after infection. Friend leukemia virus caused a rapid decrease in the number of villous cells, with a concomitant increase in the number of cells with smoother surfaces. By the end of the first 1 to 12 weeks after infection the majority of cells were smooth, many showing distinct morphologic changes, including "holes" and a spongy appearance. Nearly all of the splenocytes were abnormal in appearance by days 17 to 30 after infection, with most showing a spongy topography. These changes paralleled the marked immunosuppression induced by Friend leukemia virus infection, as well as the appearance of virus-associated surface antigen on individual splenocytes. Topographic changes evident by examination with scanning electron microscopy were not readily apparent by either standard histology or transmission electron microscopy.

Murine lymphoma-induced immunosuppression: requirement for direct tumour cell contact.

The FBL-3 lymphoma cell line caused impaired antibody formation in vivo when injected into mice intraperitoneally, and in vitro when added to normal syngeneic spleen cells immunized in vitro with sheep erythrocytes. Immunosuppression occurred only when intact viable tumor cells were cocultivated with the normal spleen cells. As few as 10(5) FBL-3 cells, when added to 5 X 10(6) normal cells, impaired antibody formation. However, cell-free extracts of filtrates from even much larger numbers of tumor cells did not affect antibody formation, either in vitro or in vivo. Heating the tumor cells at 56 degrees C or irradiation with as little as 1000 rads completely abolished immunosuppressive activity, both in vitro and in vivo. Separation of viable tumor cells from target antibody-forming cells by cell-impermeable membranes prevented immunosuppression, showing that direct cell-to-cell contact is required for immunosuppression.

Age- and sex-related differences in antibody formation and blastogenic responsiveness of splenocytes from RIII mice developing virus-induced mammary adenocarcinoma.

Inbred RIII mice, known to be infected neonatally with murine mammary tumor virus so that females develop mammary adenocarcinoma by 12-15 months of age, were examined with regard to antisheep red blood cell antibody responses at the cellular level. Female mice, 3-11 months old, compared to male mice of the same ages had consistent and significant depression of the antibody response of their splenocytes. Furthermore, female mice with adenocarcinoma showed an even greater depression of the antibody response. Spleen sizes were consistently increased in females as compared to those of male mice throughout the first year of life. The blastogenic responsiveness of the splenocytes to the B-cell mitogen Escherichia coli lipopolysaccharide and the T-cell mitogen phytohemagglutinin-P was not significantly different between male and female mice during the same periods, although the responses of the older tumor-bearing female mice to phytohemagglutinin-P were lower than those of non-tumor-bearing female mice. A complex relationship between age, sex, and immune responsiveness was evident in these mammary tumor virus-infected mice, which made it difficult to attribute a specific immune event to emergence of the mammary adenocarcinomas in the female as compared to male mice.

Immunosuppression induced in vitro by cell-free extracts of Friend leukemia virusinfected splenocytes.

The immunologic responsiveness by normal BALB/c spleen lymphocytes immunized in vitro with sheep erythrocytes was markedly suppressed by calrified cell-free homogenates of Friend leukemia virus (FLV)-infected mouse spleens. Suppression was achieved with a 3,000Xg supernatant of FLV-containing homogenates freed of cellular debris but not with crude unclarified homogenates or pelleted material after centrifugation. The immunosuppressive effects of the virus were dose dependent, whether the virus was added directly to the target spleen cells or separated from them by a 0.4mu Nuclepore filter. Suppression was prevented by heating of the virus at 100 degrees C for 10 minutes or by neutralization with antiserum to FLV. Addition of the virus as late as 48 hours after in vitro immunization of splenocytes affected the immune response. However, suppression was maximum when the clarified virus was added to the splenocytes 1 or 2 days before immunization. The agent in the FLV-infected spleen homogenates responsible for immunosuppression appeared to be the virus per se; however, virus-associated soluble factor(s) might have been involved.

Suppression of natural killer cell activity by Friend murine leukemia virus.

BALB/c mice infected with Friend murine leukemia virus (F-MuLV) evinced a decreased natural killer (NK) cell activity to susceptible target cells. This suppression increased as the interval between infection and assay was lengthened. The decrease in NK activity due to F-MuLV infection was partially reversible when spleen cells were pretreated with interferon before the cytolytic assay. The ability of F-MuLV-infected splenocytes to bind to target cells was unaltered, indicating that the defect was in the lytic phase of NK cytolysis. When mixed with uninfected spleen cells, F-MuLV-infected splenocytes suppressed their NK cell activity. This suppression was associated with a nylon wool-adherent cell population in the F-MuLV-infected spleens.

Suppression of human macrophage function in vitro by delta 9-tetrahydrocannabinol.

The ability of macrophages to function in the presence of delta 9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was evaluated. THC added to macrophage cultures prepared from human peripheral blood inhibited macrophage spreading and phagocytosis of yeast. The effects of THC were concentration dependent, with inhibitory effects observed from 10 to 1 micrograms/ml or lower. These results suggest that macrophages are more sensitive to THC than are lymphocytes because macrophage functions were inhibited by THC at concentrations that did not affect lymphocyte function. Thus, inhibition of lymphocyte function(s) by THC could be attributed to a direct effect of the drug on macrophages which indirectly results in lowered lymphoid cell activity.

Poverty and obesity: the role of energy density and energy costs.

Many health disparities in the United States are linked to inequalities in education and income. This review focuses on the relation between obesity and diet quality, dietary energy density, and energy costs. Evidence is provided to support the following points. First, the highest rates of obesity occur among population groups with the highest poverty rates and the least education. Second, there is an inverse relation between energy density (MJ/kg) and energy cost (US dollars/MJ), such that energy-dense foods composed of refined grains, added sugars, or fats may represent the lowest-cost option to the consumer. Third, the high energy density and palatability of sweets and fats are associated with higher energy intakes, at least in clinical and laboratory studies. Fourth, poverty and food insecurity are associated with lower food expenditures, low fruit and vegetable consumption, and lower-quality diets. A reduction in diet costs in linear programming models leads to high-fat, energy-dense diets that are similar in composition to those consumed by low-income groups. Such diets are more affordable than are prudent diets based on lean meats, fish, fresh vegetables, and fruit. The association between poverty and obesity may be mediated, in part, by the low cost of energy-dense foods and may be reinforced by the high palatability of sugar and fat. This economic framework provides an explanation for the observed links between socioeconomic variables and obesity when taste, dietary energy density, and diet costs are used as intervening variables. More and more Americans are becoming overweight and obese while consuming more added sugars and fats and spending a lower percentage of their disposable income on food.

Marijuana, immunity and infection.

The influence of marijuana cannabinoids on immune function has been examined extensively over the last 25 yr. Various experimental models have been used employing drug-abusing human subjects, experimental animals exposed to marijuana smoke or injected with cannabinoids, and in vitro models employing immune cell cultures treated with various cannabinoids. For the most part, these studies suggest that cannabinoids modulate the function of T and B lymphocytes as well as NK cells and macrophages. In addition to studies examining cannabinoid effects on immune cell function, other reports have documented that these substances modulate host resistance to various infectious agents. Viruses such as herpes simplex virus and murine retrovirus have been studied as well as bacterial agents such as members of the genera Staphylococcus, Listeria, Treponema, and Legionella. These studies suggest that cannabinoids modulate host resistance, especially the secondary immune response. Finally, a third major area of host immunity and cannabinoids is that involving drug effects on the cytokine network. Employing in vivo and in vitro models, it has been determined that cannabinoids modulate the production and function of acute phase and immune cytokines as well as modulate the activity of network cells such as macrophages and T helper cells, Th1 and Th2. These results are intriguing and demonstrate that under certain conditions, cannabinoids can be immunomodulatory and enhance the disease process. However, more studies are needed to determine both the health risk of marijuana abuse and the role of the cannabinoid receptor/ligand system in immune regulation and homeostasis.

Acid sensitivity of cell-free and cell-associated HIV-1: clinical implications.

Infectivity of free and cell-associated human immunodeficiency virus type 1 (HIV-1) treated in vitro at pH 7.4 to 4.9 for 2 hours was assessed on susceptible CEM-ss cells. Viral activity was monitored by cytopathology and production of reverse transcriptase and p24 antigen. The infectivity of cell-free virus was gradually inactivated and at pH 5.4 was completely lost, with or without subsequent adjustment of pH to neutral. Virus-producing cells also gradually lost their ability to infect as the pH decreased; however, restoration of neutral pH resulted in regained infectivity. Since the pH values used in the study are similar to those found at various entry sites of the human body, the data may be relevant to the mode of transmittal of HIV.

Suppression by delta-9-tetrahydrocannabinol of lipopolysaccharide-induced and intrinsic tyrosine phosphorylation and protein expression in mouse peritoneal macrophages.

Lipopolysaccharide (LPS, 100 ng/mL)-induced tyrosine phosphorylation of four proteins (p41, p42, p77, and p82) in mouse resident peritoneal macrophages was observed using a monoclonal anti-phosphotyrosine antibody PY20 immunoblotting method. Macrophages pretreated for 3 hr with 1 microgram delta-9-tetrahydrocannabinol (THC)/mL had decreased tyrosine phosphorylation of p77 and p82 after incubation with LPS for 30 min. Simultaneous treatment of macrophages with THC (10 micrograms/mL) plus LPS for 30 min had a similar effect on p77 and p82 tyrosine phosphorylation. When the THC pretreatment protocol was combined with the simultaneous treatment protocol, 0.5 and 5 micrograms THC/mL, respectively, completely blocked LPS-induced p77 and p82 tyrosine phosphorylation. However, neither simultaneous treatment with THC nor pre- and simultaneous treatment had any effect on LPS-induced tyrosine phosphorylation of p41 and p42 in macrophages. Pretreatment with 1 microgram THC/mL followed by simultaneous treatment with 10 micrograms THC/mL induced a p43 protein that showed tyrosine phosphorylation in place of p41 and p42. Further analysis of THC effects on macrophages revealed an increase in tyrosine phosphorylation as an immediate early even after THC treatment. Prolonged treatment of macrophages with THC resulted in a broad suppression of tyrosine phosphorylation and some cellular protein expression. Three cellular proteins (p65, p70, and p72) seemed most susceptible to inhibition by THC. The data suggest that suppression of tyrosine phosphorylation by THC in macrophages may be one of the mechanisms associated with inhibition of cell function, including the suppression of tumor necrosis factor-alpha release from macrophages.

Delta-9-tetrahydrocannabinol: an inhibitor of STAT1 alpha protein tyrosine phosphorylation.

Tyrosine-phosphorylated signal transducer and activator of transcription 1 alpha (STAT1 alpha) is a 91-kDa protein responsible for interferon-gamma (IFN-gamma)-dependent transcription. The present study demonstrates that activation by IFN-gamma of murine macrophages resulted in tyrosine phosphorylation of STAT1 alpha identified by immunoprecipitation. The tyrosine phosphorylation of STAT1 alpha was found highly sensitive to treatment by delta-9 tetrahydrocannabinol (THC), a major marijuana component. Subsequently, the isoform formation of p91 due to tyrosine phosphorylation was reduced in THC-treated macrophages. Although inhibition by THC of the tyrosine phosphorylation of STAT1 alpha induced by IFN-gamma was in a THC concentration-related manner, the tyrosine phosphorylation of other proteins induced by lipopolysaccharide/IFN-gamma treatment of macrophages appeared insensitive to THC treatment. Our data suggest that blockade by THC of tyrosine phosphorylation of STAT1 alpha may be an important mechanism involved in the broad immunosuppressive effects of THC.

Epidemiologic factors correlated with multiple sexual partners among women receiving prenatal care.

A cross-sectional survey methodology was used to respond to the need of a local health department to identify correlates of high-risk behaviors related to human immunodeficiency virus (HIV) transmission among pregnant women attending prenatal care clinics. This study of 488 maternity patients was conducted at two public health clients in Tampa, Florida, in 1991. The prevalence of high-risk behaviors was assessed using a self-administered questionnaire. Out of 428 respondents, 25% reported having had intercourse with two or more male partners in the past year. Following multiple logistic regression analysis, four variables remained consistently and significantly associated (P < 0.05) with having two or more sexual partners: (1) annual household income of less than $10,000/y (prevalence odds ratio (POR) = 4.5; 95% confidence limits (CL): 1.5, 13.1); (2) history of prostitution (POR = 8.1; 95% CL: 1.5, 42.1); (3) history of rape or forcible intercourse (POR = 2.2; 95% CL: 1.0, 4.6); and (4) an expressed desire for confidentiality among women seeking further information about acquired immunodeficiency syndrome (AIDS) prevention (POR = 2.1; 95% CL: 1.1, 4.0). Assessment of these factors may lead to better direction of HIV education programs, as well as identification and counseling of specific individuals at high risk for engaging in behaviors that can lead to HIV infection. Short, self-administered questionnaires provide confidential, rapid, and inexpensive means of generating baseline data for further interventions.

A 7-kda protein encoded by the bamhi-h gene family of mareks-disease virus is produced in lytically and latently infected-cells.

Marek's disease virus (MDV) BamHI-H gene family was transcribed specifically in cells infected with oncogenic MDV, and the transcripts were prematurely terminated in cells infected with non-oncogenic attenuated strains of MDV due to the amplification of a 132 bp repeat located downstream of their promoter. There are six small open reading frames in the two differently spliced and two unspliced transcripts. This report describes expression of BHa gene open reading frame A, present in 1.7 kb unspliced transcript. Antibody was raised against BHa C-terminal polypeptide. The antibody showed specific reaction to the GST fusion protein derived from BHa protein. Immunoprecipitation with the lysates of infected cells was carried out. The results indicated that 7 kDa BHa protein was immunoprecipitated from the lysates of oncogenic MDV infected chicken embryo fibroblasts (CEF) and a MSB-1 lymphoblastoid cell line derived from Marek's disease tumor but not from non-oncogenic MDV infected CEF or uninfected CEF. 36 kDa protein was co-immunoprecipitated with 7 kDa BHa protein in oncogenic MDV infected CEF but was not detected in MSB-1 cells. This is the first report to show that a small open reading frame of the BamHI-H gene family was in fact expressed in MDV infected cells.

Prolonged proliferation of primary chicken-embryo fibroblasts transfected with cdnas from the bamhi-h gene family of mareks-disease virus.

Oncogenic virus-specific 1.69 kb and 1.5 kb cDNAs, derived from the tumorigenicity-associated BamHI-H gene family of Marek's disease virus (MDV), were cloned into the eukaryotic expression vector (PRC/CMV) and designated as PRC/CMV-1.69 and PRC/CMV-1.5, respectively. Chicken embryo fibroblasts (CEFs) transfected with PRC/CMV-1.69 or PRC/CMV-1.5 plasmid DNA showed reduced serum-dependence for growth compared with PRC/CMV vector-transfected CEFs. Also, PRC/CMV-1.69 or PRC/CMV-1.5 DNA transfected CEFs proliferated for a longer period of time (20-22 passages) compared with the CEFs transfected with PRC/CMV plasmid DNA (10 passages). Reduced serum-dependence for growth and prolonged proliferation of CEFs after in vitro transfection with PRC/CMV-1.69 or PRC/CMV-1.5 indicated that these two cDNAs stimulate cell growth. These data indicated that one of the functions of the BamHI-H gene family of MDV is cell-growth control, which may play an important role in the establishment and maintenance of uncontrolled growth of tumor cells induced by MDV.

Morphine effects on HTLV-I infection in the presence or absence of concurrent HIV-1 infection.

Human T-cell leukemia virus type I (HTLV-I) infection is emerging as an important complication in HIV infection and AIDS in injecting drug users. HIV-1 and HTLV-I share a common host in CD4+ T lymphocytes. However, the result of HIV-1 infection is the decimation of this cell population, whereas a hallmark of HTLV-I infection is the inappropriate proliferation of infected cells. Combined epidemiologic data suggest that HTLV-I infection is enhanced during concurrent HIV-1/HTLV-I infection; however, there are currently no in vitro studies focusing on the effects of drugs of abuse on retrovirus coinfection. We have found that in an in vitro coinfection system (HIV-1 + HTLV-I), morphine treatment further enhanced the levels of HTLV-I p19. In addition, indicators of in vitro infection by cell-free HIV-1 were reduced by morphine treatment in both single and dual in vitro infection experiments. Interleukin 2 levels in the affected cultures were found to increase with combined HTLV-I infection and morphine treatment. These in vitro results indicate the need to further explore the activity of HTLV-I within opiate-treated cells, as this oncoretrovirus appears to be especially sensitive to morphine-induced alterations to its host cell environment.

Modulation of ex vivo cytokine production by splenocytes using in vitro combined therapeutics in murine retroviral model.

Altered levels of Type 1 and Type 2 cytokines are important in retrovirus-induced immunosuppression. The combination of immunostimulatory agents with antiviral drugs alters the course of murine retroviral infections. Previously, it was demonstrated that in vitro treatment of noninfected splenocytes and in vivo treatment of Friend leukemia virus (FLV)-infected mice with the combination of azidothymidine (AZT) and methionine enkephalin (MENK) significantly increases Type 1 cytokine levels and decreases Type 2 cytokines compared with treatment with only AZT. In order to study the effect of the time of initiation of immunomodulation on the course of retroviral infections, we examined the kinetics of cytokine production by isolated splenocytes from infected mice. BALB/c mice were infected with FLV, and spleen cells were removed at specified times postinfection (days 1, 3, 7, 10, and 14). Interleukin (IL)-2, interferon (IFN-gamma, IL-4, and IL-10 production by unstimulated or ConA-stimulated splenocytes treated in vitro with AZT, MENK, or AZT + MENK was determined after 48 h. The capacity of the isolated splenocytes to produce the Type 1 cytokines IL-2 and IFN-gamma in response to stimulation with ConA and combination therapy decreased over the course of infection. These results suggest that MENK treatment initiated later in the course of infection is unable to modulate the cytokine profile and would likely be ineffective in altering the course of FLV induced-disease. The results indicate the necessity to initiate antiretroviral therapy early in infection. Such information may be applicable in designing future regimens for HIV-1 infections in humans.

Factors from lymphoid cell tumor affecting immune responses.

Friend leukemia virus induces erythroblastic leukemia in genetically susceptible BALB/c mice. FLV-containing leukemic cells markedly depressed the humoral immune response to SRBC in the appropriate mouse strain. Both immunosuppression and leukemogenesis were readily transmitted by cell-free virus-containing homogenates of the FLV leukemic splenocytes into normal BALB/c mice. In the present study it was found that both Friend leukemic splenocytes as well as virus containing extracts from the leukemic cells were neutralized by heating and by specific antisera. Suppressive activity passed through a 0.45 mu filter but not a 300,000 MW filter and could be pelleted at 100,000 x g. They were also highly resistant to inactivation by irradiation. Mice given leukemic splenocytes after irradiation with up to 32.000 rads still developed leukemia. Addition of either normal or irradiated FLV-leukemic cells to normal spleen cell cultures in vitro markedly suppressed antibody formation. At least 32,000 rads were required to significantly impair the immuno-suppressive activity of the FLV-leukemic cells. Thus, virus per se appears to be directly responsible for suppression of antibody formation to FLV.

Reducing ice cream energy density does not condition decreased acceptance or engender compensation following repeated exposure.

OBJECTIVES: The preferences for high-fat foods are believed to be based on their sensory attributes and energy density; however less is known about how such preferences might be weakened, other than in response to deterioration in flavor or textural quality. The aim of the present study was to see whether acceptability of reduced fat/energy foods would wane as the original post-ingestive nutritional benefits are reduced when palatability remains essentially constant. DESIGN: Repeated measures, within-subjects design conducted in two counterbalanced three week trials. SETTING/SUBJECTS: Sixteen normal-weight males (mean age 25.8 +/- 1.2 y) came to our laboratory at the Hôpital Hotel Dieu in Paris to eat an afternoon snack on 13 consecutive days (excluding weekends). INTERVENTION/OUTCOME MEASURES: Intake was recorded following repeated exposure to two flavors of standard (10% fat as a percentage of total solids weight), and low (3%) fat ice cream. One group received standard vanilla or low-fat strawberry ice cream on alternate days for two consecutive weeks; these flavor associations were reversed for a second group. The two flavors were rated as equipalatable at the beginning of the experiment at all energy levels. RESULTS: Subjects consumed the same quantity of ice cream throughout the experimental period, independent of energy density or flavor. Consequently, aggregate (summed) energy intake for subjects consuming low-fat ice cream was significantly lower (by 581 kJ (139 kcal), 15.4 g fat). Food intake records for the 24 h period immediately following the test sessions revealed no compensation for fat or energy. Despite the 28% reduction in energy density for the low-fat version, acceptance for the flavors associated with the reduced-energy versions had not declined by the end of the experimental period. CONCLUSIONS: The findings suggest that acceptance of reduced-fat foods may not be critically dependent on the post-ingestive metabolic effects when the reductions in energy density are small. Further tests with more severe reductions, and perhaps over more prolonged time periods, will be necessary to determine at what level of substitution acceptance might begin to deteriorate.

Enhancement of plasma levels of tumor necrosis factor alpha but not interleukin-6 following endotoxin injection of Friend leukemia virus infected mice.

Friend leukemia virus complex (FLC) infection of BALB/c mice causes a rapid, progressive suppression of most immune functions. In the present study, FLC infection resulted in increased induction by bacterial lipopolysaccharide (LPS) of tumor necrosis factor alpha (TNF alpha) but not IL-6. TNF alpha levels were significantly elevated beginning 11 days post infection and increasing levels were measured through day 21. The highest TNF alpha levels in FCL-infected mice were as much as 100-fold higher than in LPS treated non-infected mice. Peak plasma levels of TNF alpha were seen between 1 and 2 hr after LPS induction, as compared to a peak at 1 hr in controls. The ability of LPS to stimulate TNF alpha was concentration dependent over a range of 0.005 to 50 micrograms per mouse. Using anti-TNF alpha antiserum, cytotoxic activity of plasma was shown to be due specifically to TNF alpha. These data suggest that induction of TNF alpha and IL-6 is regulated by different mechanisms in FLC-infected mice.

Combined in vitro effect of marijuana and retrovirus on the activity of mouse natural killer cells.

Both marijuana and retroviruses impair natural killer (NK) cell functions. No data on their simulataneous effects are available. Similarities to human AIDS induced early by Friend leukemia complex (FLC) and its replication competent helper Rowson-Parr virus (RPV) provides a mouse model to study drug-virus action. Leukemia susceptible BALB/c and resistant C57BL/6 mice were infected, then at time intervals their nylon wool-separated splenocytes were exposed to tetrahydrocannabinol (THC) for 3h. Natural killer (NK) cell activity against Yac-1 cells was assayed by 51Cr-release for 4 and 18h. Recovery of splenocytes was found to be suppressed by FLC, but in BALB/c only by RPV. After a transient enhancement in C57BL/6 by FLC, NK cell activity of both mice became suppressed early (2 to 4 days), normalized subsequently and enhanced late (11 to 14 days) postinfection. A moderate increase in BALB/c, no change in C57BL/6 were induced by low (1-2.5 microgram/ml) THC doses. NK cell activity of BALB/c became suppressed exponentially by higher (5-10 microgrtam/ ml) THC doses in 18h as compared to 4h assays, while its proportional and moderate impairment was seen in C57BL/6. The magnitude of NK cell activity of infected mice was determined by THC: enhancement or impairment followed those of untreated, infected counterparts, but on the level of THC-treated cells. Low doses hardly, high doses additively influenced NK cells of infected BALB/c. THC hardly affected very early and late enhancement in NK cell activiy of FLC infected C57BL/6, but augmented RPV induced suppression late in 18h assays. Genetic factors similar to endotoxin resistance, altered cytokine profile might determine these effects. Similar phenomena in humans might result in earlier manifestation of AIDS.