Cocaine and sigma-1 receptors modulate HIV infection, chemokine receptors, and the HPA axis in the huPBL-SCID model

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. A human xenograft model, in which human peripheral blood mononuclear cells were implanted into severe combined immunodeficiency mice (huPBL-SCID) and infected with a HIV reporter virus, was used to investigate the biological interactions between cocaine and HIV infection. Systemic administration of cocaine (5 mg/kg/d) significantly increased the percentage of HIV-infected PBL (two- to threefold) and viral load (100- to 300-fold) in huPBL-SCID mice. Despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic-pituitary-adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of 10(-8) M cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID model following systemic exposure to cocaine (54.1+/-9.4% increase over control, P<0.01). This effect preceded the boost in viral infection and waned as HIV infection progressed. Cocaine has been shown to mediate immunosuppressive effects by activating sigma-1 receptors in immune cells in vitro and in vivo. Consistent with these reports, a selective sigma-1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. Our results suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating sigma-1 receptors and by modulating the expression of HIV coreceptors.     

Humoral and cellular responses of mice to infection with a cold-adapted influenza A virus variant.

The serum antibody response and four different cellular immune responses (cytotoxic T cells, delayed-type hypersensitivity T cells, natural killer cells, and cytotoxic macrophage levels) induced in CBA/H mice were measured at different times after intranasal inoculation of a cold-adapted (ca) variant of influenza A virus, influenza virus A/Ann Arbor/6/60-ca, or the parental virus, influenza virus A/Ann Arbor/6/60. At the highest dose of virus inoculated (5 log10 50% tissue culture infective doses), all four cellular responses reached high levels in the lungs of both groups of mice, and serum antibody titers were detected on day 20 after inoculation of either virus. However, whereas extensive replication of the parental virus occurred in the mouse lungs, very limited replication of the ca variant was observed. Macroscopically, infection with the parental virus caused gross lung damage, whereas such damage was almost absent in mice inoculated with the ca variant. Inoculation of 2 to 5 log10 50% tissue culture infective doses of the parental virus induced high cytotoxic T-cell responses, whereas only the highest dose of the ca variant caused a clearly significant cytotoxic T-cell response. As an inoculum of 5 log10 50% tissue culture infective doses of the ca variant caused a substantial primary immune response without appreciable lung damage, the avirulence of the ca variant may be primarily related to its limited ability to replicate productively in mouse lungs.     

Characterization of age-related changes in natural killer cells during primary influenza infection in mice

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.     

A mouse hepatotropic variant of influenza virus

A hepatotropic variant of avian influenza virus A/Turkey/England 63 (Hav 1, Nav 3) was selected by serial passages in mouse liver. Adaptation to this organ was established after 13 in vivo passages and was found to improve during further passages as shown by increasing rates of replication in livers of ICR mice. The mutant virus finally selected was stable and differed from the original virus mainly in lethality upon intraperitoneal injection in mice, in its ability to grow to high titers in livers of susceptible animals and in plaque morphology in chick embryo fibroblasts. No differences were detected in hemagglutination inhibition and neutralization by standard mouse antisera. Pathogenicity for the liver was independent of the route of inoculation, included other laboratory animals sensitive to influenza virus and could be inhibited by amantadine. Fatal hepatitis in 50 per cent of susceptible mice by the intraperitoneal route required from 10 to 20 EID50-. Pathological changes consisted of severe necrosis of liver parenchyma accompanied by release of F antigen into the serum and were apparently due to virus replication in hepatic cells as evidenced by immunofluorescence. The main implications of this animal model for studies on experimental hepatitis and on myxovirus-host interactions in an organ not usually associated with influenza are discussed.     

Modification of spleen cell subsets by chronic cocaine administration and murine retrovirus infection in normal and protein-malnourished mice.

We have developed an experimental mouse model to study the effect of daily cocaine administration on the immune system during an acquired immune deficiency syndrome (AIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with the development of functional murine AIDS. Increasing doses of cocaine given by daily intraperitoneal injection for 11 weeks reduced body weight. A daily cocaine injection in some mice as well as a saline injection in others showed a decrease in the percentage of Thy 1.2+, CD4+ and CD8+ cells, while both treatments increased the percentage and absolute numbers of B-cells per spleen. Saline and cocaine treatment induced an increase in gamma-IFN and TNF-alpha production by splenocytes. Cocaine treatment favored a decrease in sIL-2R secretion. Saline and cocaine treatment had slightly different effects on the splenocytes of protein-malnourished mice. Cocaine treatment induced an increase in the percentage of CD8+ cells. Saline and cocaine treatments decreased the number of Mac 1+ cells in the spleen. Moreover, saline- and cocaine-treated protein-malnourished mice splenocytes did not present the increase in gamma-IFN production as well-nourished mice splenocytes showed. Retrovirus-infected mice showed a decrease in the percentage of Thy 1.2+ and CD8+ cells and an increase in the percentage and absolute numbers of CD4+, IL-2R+, Mac 1+ and B-cells. Cocaine partially prevented the enlargement of lymphoid organs due to lymphoid cell proliferation induced by murine retrovirus infection, but had little effect on the elevated percentage of CD4+ cells or B-cells or the depressed numbers of CD8+ cells associated with virus infection. However, cocaine did reduce the number of activated IL-2R+ cells and macrophages (Mac 1+) in addition to reducing the total number of cells per spleen in all subsets in retrovirus-infected mice, but not in uninfected controls. Cocaine treatment and retrovirus infection alone or in combination suppressed the release of sIL-2R into supernatant fluid during in vitro culture of splenocytes. These data illustrate that cocaine treatment modulates cell proliferation in retrovirus-infected mice and thus modifies the absolute number of cells in those subsets already altered by retrovirus infection. Retrovirus-infected and retrovirus-infected cocaine-treated protein-malnourished mice showed similar results.     

Antiviral activity of a synthetic analog of prostaglandin A in mice infected with influenza A virus

Summary We have previously shown that prostaglandins of the A series potently inhibit virus replication in several virus-host systems in vitro. In the present report we have studied the effect of a long-acting synthetic analog of PGA, 16,16-dimethyl-PGA₂ (Di-M-PGA₂), on virus infection in vivo, using as a model Balb/c mice infected with influenza A (PR8) virus. Depending upon the dose of viral inoculum, PR8 virus caused the death of 50 to 100% of the animals in a period of 8–20 days. Di-M-PGA₂-treatment significantly increased mouse survival by an average of 40%, independently of the dose of inoculum and the age of the animals. The fact that Di-M-PGA₂-treatment decreased virus titers in the lungs and did not alter the host immune response, suggested that PGA's therapeutic action was due to suppression of virus replication. Finally, two anti-inflammatory compounds, which inhibit prostaglandin synthesis, aspirin and indomethacin, were shown not to significantly alter mouse survival in this system.     

Alteration of thymic cell subsets by cocaine administration and murine retrovirus infection in protein undernourished mice.

Retrovirus infection, cocaine administration, and nutritional deficiencies are known to individually produce impairment of the immune system. Therefore, we developed a murine model to study the effect of daily cocaine administration, protein malnutrition, and retrovirus infection causing murine AIDS on the lymphoid cell populations of the thymus. C57BL/6 female mice fed a diet containing 4% protein were studied following chronic cocaine administration and LP-BM5 murine leukemia virus (MuLV) infection. Cocaine administration reduced body and thymus weight. Cocaine partially prevented thymus enlargement due to lymphoid cell proliferation induced by murine retrovirus infection. Cocaine treatment affected dramatically the thymus of protein-malnourished mice where the absolute number of Thy 1.2+, CD4+ and CD8+ cells represented only 10% of the control values. Daily saline injection also induced a significant decrease in the number of Thy 1.2+, CD4+ and CD8+ cells per thymus. These results suggest that the thymus glands of mice fed a low protein diet were susceptible to stress. Retrovirus infection provoked a decrease in the percentage and absolute number of Thy 1.2+, CD4+ and CD8+ cells in the thymus. This effect was potentiated by cocaine treatment. Therefore, cocaine was able to potentiate the impairment of the immune system caused by MuLV infection. We consider that cocaine could alter the immune system by altering the expression of T cell differentiation markers after direct interaction with thymocytes or through the neuroendocrine-thymus axis. Moreover, this effect was more dramatic and severe during protein malnutrition.     

Virus infection-associated bone marrow B cell depletion and impairment of humoral immunity to heterologous infection mediated by TNF-alpha/LTalpha

We previously showed that influenza virus infection of mice induces a depletion of bone marrow B lineage cells due to apoptosis of early B cells mediated by a mechanism involving TNF-alpha/LTalpha. Here we demonstrate that this effect is also observed with acute lymphocytic choriomeningitis virus (LCMV) infection and resulted in a deficiency of both splenic transitional B cells and mature follicular B cells. To determine whether there was an associated impairment of humoral immunity, we infected mice with LCMV and 10 days later at the peak of the B cell depletion, inoculated them with influenza virus. We found that influenza virus-specific antibody titers were dramatically reduced in mice recovering from LCMV infection compared to those in mice infected with influenza virus alone. Further, we showed that there was no reduction of the influenza virus-specific antibody response in LCMV-infected TNF-alpha/LTalpha-deficient mice, suggesting that TNF-alpha/LTalpha-mediated effects on bone marrow and/or peripheral lymphocytes were responsible for the observed impairment in humoral immunity. These results show that the TNF-alpha/LTalpha production induced following infection with diverse viruses has detrimental effects on early B cells in the bone marrow, and may be among the factors that lead to the severely compromised humoral immunity observed to subsequent heterologous infections.     

The influence of endogenous hypocorticism on the replication of influenza virus in the mouse lungs and the production of specific antibodies

The replication of pathogenic influenza virus A/PR/8/34 in the lungs and the synthesis of virus-neutralizing (VN) and haemagglutination-inhibition (HI) antibodies has been studied in mice with endogenous hypocorticism induced by a bilateral adrenalectomy. The adrenalectomized mice appeared to be more susceptible to influenza infection as compared to the mock-operated ones. This was evident from earlier deaths and higher death rate in mice inoculated with 50 EID50, 1000 EID50, and 6000 EID50 of the virus, respectively. A tendency towards decrease of specific antibody titres and the resistance to reinfection with influenza virus A/PR/8/34 was also observed.     

The influence of glucocorticoid immunosuppression on the course of experimental influenza pneumonia

The study demonstrates the effects of kenalog (Kn), a synthetic glucocorticoid hormone, on the course of virus A/Aichi/2/68 influenza in white mice. In doses of 5 and 10 mg/kg, Kn reduced the weight of the adrenal glands, thymus and spleen, which was accompanied by decrease of the resistance to the mentioned virus, judging by LD50 decrease vs. this index in the control infected group. Besides, four days after infecting with 5 LD50 of influenza virus (IV), lung virus and interferon titers were significantly lower in mice pretreated with Kn vs. mice treated with placebo. Lung cell susceptibility to IV in vitro was identical in mice treated with Kn or placebo. In ultrathin lung sections of IV-infected mice, both experimental and control ones, there was virus budding in bronchial epithelium cells and type I and II alveolocytes. Analysis of inflammatory effusion compound in semithin lung sections 6 days after IV infection, found a substantially smaller number of mature alveolar macrophages (AM) and a bigger number of neutrophiles vs. infected controls. The authors reckon that higher mortality of mice pretreated with Kn before infecting, is caused not by enhancement of IV reproduction in target lung cells during influenza development, but by the contribution of other pathogenic factors. One of those may be increase of neutrophilic migration into the lungs; neutrophiles are more able to realize their significant destructive potential under the condition of reduction in the clearing function of AM and IV infection.     

Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS.

LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C57BL/6 female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LP-BM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CD8+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8+ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS.     

Influenza virus infection of hamsters. A model for evaluating antiviral drugs

Summary Inoculation of hamsters with influenza virus [A/PR/8/34HON 1] produces an inapparent infection which can be monitored by virus titrations of nasal washes or of homogenates prepared from trachea or lung. Antibody can be detected in the serum within 7 days following virus inoculation. Hamsters previously infected were found to be resistant to challenge with the same virus. The utility of this model for evaluating anti-influenza drugs was demonstrated with two compounds. Calcium elenolate, a virucidal agent, reduced the virus titers of nasal washes when the drug was given as nose drops near the time of virus inoculation so as to affect high drug concentrations in the nasal passages. Virazole, an inhibitor of virus replication, reduced the virus titers of the nasal washes when multiple drug treatments were given as nose drops in an effort to provide drug during the time of virus replication. The model described may provide a useful means of evaluating potential antiviral drug candidates inasmuch as the drug can be delivered directly into the nasal passages in a non-fatal influenza infection in a convenient laboratory animal.With 1 Figure     

Effect of liposomal beta-carotene on experimentally lethal influenza infection]

Effect of beta-carotene on experimental infection with A/Aichi/2/68 (H3B2) is studied in mice infected in an infective dose of 5-10 LD50. The drug notably decreased the mortality in experimental group in comparison with the control. Disease symptoms were less expressed and deaths observed later (7 days vs. 4 in the control) in mice treated with beta-carotene. Histological analysis of the lungs showed smaller foci of lesions. Metaplastic changes in the bronchial epithelium, typical of late terms of infection, were far less expressed in these animals. On the other hand, virus titers decreased negligibly in comparison with the control group. Electron-microscopic study of the effect of beta-carotene on virus population showed that virus replication in chick embryos in the presence of beta-carotene led to an increase in the percentage of filamentous and giant polygenome virus particles. The data indicate that beta-carotene is a promising drug for prevention and treatment of influenza.     

Enumeration and characterization of virus-specific B cells by multicolor flow cytometry

To better characterize B cell responses induced to influenza virus, we developed an assay to directly quantify and characterize virus-specific B cells. We used purified and biotinylated whole virus as well as the major influenza virus surface antigen, hemagglutinin (HA) to label virus-specific B cells induced by immunization of mice with whole influenza virus in adjuvant. Immunization with adjuvant alone caused non-specific binding of whole virus to a large number of B cells in the draining lymph nodes as assessed by flow cytometry. This precluded the use of whole virus as a specific staining reagent. In contrast, staining with bromelain-cleaved purified and biotinylated influenza virus HA identified a small population of B cells (roughly 1%) only in the draining lymph nodes of virus-immunized mice. FACS-purification and subsequent ELISPOT analysis showed that HA-labeled B cells contained the vast majority of virus-specific antibody-secreting cells at day 10 after immunization. Overall, virus-specific antibody-secreting cells comprised roughly 10% of the HA-labeled cells. Using HA-staining in conjunction with 8-color flow cytometry we further demonstrated that close to 90% of the HA-labeled cells were CD19+ IgD- CD23- CD24high CD38low germinal center B cells, many of which had incorporated bromodeoxyuridine, indicating recent cell division in vivo. We conclude that viral HA can be used in conjunction with cell surface and intracytoplasmic stains in multicolor flow cytometry to provide detailed phenotypic and functional information on virus HA-specific B cells.     

Stimulation of mouse macrophage antigen presentation by cocaine

Cocaine has been demonstrated to have multiple effects on the immune system. Here, we determined the effects of cocaine on macrophage antigen presentation, using an in vitro antigen presentation assay after macrophages were treated with cocaine both in vitro and in vivo. Our results showed that in vitro treatment of macrophages with cocaine significantly enhanced macrophage's ability to present ovalbumin (OVA) and the enhancement was also demonstrated in the macrophages of cocaine-injected mice. The presentation of an OVA-derived antigenic peptide (OVA_323-339), however, was not affected. In vitro cocaine treatment neither affected antigen uptake nor major histocompatibility complex (MHC) II expression and the expression of co-stimulatory molecules B7. These results suggest that cocaine may act on an early event in the antigen handling by accessory cells.     

Anti-influenza-virus activity of total alkaloids from <Emphasis Type="Italic">Commelina communis</Emphasis> L.

The antiviral activity of total alkaloids from Commelina communis L. (TAC) against influenza virus A/PR/8/34 (H1N1) was investigated in vitro and in vivo. TAC exhibited an inhibitory action on the growth of influenza virus in Madin-Darby canine kidney cells when added before or after viral infection. In mice infected with influenza virus, orally administered TAC at 8, 16 or 32 mg/kg per day for 6 days significantly increased the survival rate, prolonged the mean survival time and reduced the viral titers in the lung and the lung index, compared with that of the untreated virus control. The results obtained suggest that TAC has a pronounced protective effect against infection by influenza A virus.     

Mycoplasma-Virus Interrelationships in Mouse Tracheal Organ Cultures

The effects produced by single and mixed infections with Mycoplasma pulmonis and influenza A/PR-8 virus were studied in mouse tracheal organ cultures. M. pulmonis multiplied in the tracheal organ cultures, producing inhibition of ciliary activity and histologic tissue damage. The organism grew in close association with the cell membranes but did not appear to attach directly to the membranes or the cilia. Influenza A virus also replicated in tracheal organ cultures, producing ciliary inhibition and more extensive cytopathologic changes. Virus particles were seen by electron microscopy to attach to and cause clumping of the cilia. Simultaneous infection of the organ cultures with mycoplasma and virus resulted in more rapid inactivation of ciliary activity and greater tissue damage than occurred when the cultures were infected with only mycoplasma or virus. Presence of the virus appeared to have no effect on the growth of the mycoplasma; however, the mycoplasma partially interfered with virus replication.     

Cross-protection against influenza virus infection by intranasal administration of M2-based vaccine with chitosan as an adjuvant

Influenza vaccines based on conserved antigens could provide cross-protection against infections by multiple subtypes of influenza A virus. Influenza matrix protein 2 (M2) is highly conserved in all influenza A strains. In this study, we deleted the transmembrane domain of the M2 of the avian influenza virus (AIV) A/Chicken/Jiangsu/7/2002 (H9N2) strain to create an M2 without a transmembrane domain, named sM2, which was efficiently expressed in Escherichia coli. The sM2 protein was administered intranasally to mice in combination with chitosan adjuvant three times at an interval of 3 weeks. Three weeks after the last immunization, the mice were challenged with a lethal dose (5 × LD₅₀) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, body weight, and the serum antibody titers of the mice. Nasal administration of 15 μg sM2 in combination with chitosan completely protected mice against the homologous virus and protected 90 and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the sM2 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and that the adjuvant chitosan improved the efficacy of the sM2 vaccine.