Effect of bacterial or porcine lipase with low- or high-fat diets on nutrient absorption in pancreatic-insufficient dogs

The neurotoxic effects of the feline immunodeficiency virus (FIV) and FIV envelope proteins were measured in primary cultures of feline cortical neurons. Envelope protein from the FIV-PPR strain promoted neuronal swelling and death, whereas envelope protein from the FIV-34TF10 isolate produced intermediate or negligible toxicity. No effect was observed in control cultures treated with envelope protein from the Epstein-Barr virus. A concentration-effect curve showed that FIV-PPR protein produced maximal toxicity at 200 pM protein and decreased toxicity at higher concentrations, which is consistent with previous reports of the HIV-1 surface glycoprotein, gp120. These effects required the presence of low concentrations of glutamate. Using the natural host cells as targets, the effects of envelope protein and infectious virions were directly compared. All of the toxic activity could be attributed to non-infectious interactions between the viral envelope and target cells. Addition of 1 microM tetrodotoxin failed to block the effects of FIV-PPR in the presence of 20 microM glutamate. Toxicity would appear to involve two steps in which the envelope protein first sensitizes neurons through non-synaptic interactions (TTX insensitive) thereby setting the stage for enhanced synaptic activation via glutamate receptors (TTX sensitive).     

CXCR4 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.     

Elevated interleukin-10-to-interleukin-12 ratio in feline immunodeficiency virus-infected cats predicts loss of type 1 immunity to Toxoplasma gondii

Similar to human immunodeficiency virus, feline immunodeficiency virus (FIV) induces immunodeficiency and enhanced susceptibility to secondary pathogens. To explore cytokine alterations in lentivirus immunodeficiency, constitutive mRNA expression was measured in lymph nodes of healthy and FIV-infected cats before and after challenge with Toxoplasma gondii. Cytokine mRNA expression was similar in control and FIV-infected cats during the first 10 weeks after infection. At 16 weeks, interferon (IFN)-gamma, tumor necrosis factor-alpha, and interleukin (IL)-10 mRNA were increased in FIV-infected cats. Challenge with T. gondii induced an increase in IL-2, IFN-gamma, and IL-12 in the lymph nodes of control cats, whereas IFN-gamma and IL-10 but not IL-2 or IL-12 increased in the lymph nodes of FIV-T. gondii coinfected cats. These results indicate that FIV immunodeficiency may derive from a failure to generate an IL-12-dependent type 1 response and that an elevated level of IL-10 mRNA expression is a predictor of lentivirus immunodeficiency.     

Point-of-care (POC) measurement of coagulation after cardiac surgery

Infection of feline immunodeficiency virus (FIV) has been shown to induce apoptosis that might be associated with the lymphocyte depletion in the infected cats. To investigate the inhibitory effect of antioxidants on FIV-induced apoptosis, we examined the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on apoptosis and virus replication in feline lymphoblastoid (Fel-039) and fibroblastoid (CRFK) cell lines infected with FIV. The treatment with NAC or AA induced a significant inhibition of viral replication and apoptosis in Fel-039 cells and tumor necrosis factor alpha (TNF-alpha)-treated CRFK cells infected with FIV. Both cell lines in the presence of noncytotoxic concentrations of NAC or AA showed in increase of intracellular glutathione (GSH) level, which might protect the cells against oxidative stresses exerted by FIV infection and TNF-alpha treatment. On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH level might be effective for inhibition of viral replication and cell death associated with the development of immunodeficiency.     

The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.     

Cytokine expression and tumorigenicity of large granular lymphocytic leukemia cells from mice transgenic for the tax gene of human T-cell leukemia virus type I

The human T-cell leukemia virus type I (HTLV-I) regulatory protein, Tax, has been speculated to play a major role in HTLV-I leukemogenesis. Indeed, several studies have suggested that upregulation of various cellular oncogenes and cytokines by Tax may explain the pathogenesis observed in HTLV-I-infected individuals, as well as several Tax-transgenic animal models. We report here the analysis of cytokine expression in a Tax-transgenic animal model with large granular lymphocytic (LGL) leukemia. Two different transgenic mice showed identical expression of interleukin-1alpha (IL-1alpha), IL-1beta, interferon gamma (IFNgamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in peripheral tail tumors. Interestingly, LGL cell lines derived from these same tumors expressed high levels of both IFNgamma and GM-CSF, which correlated with the level of Tax expression. These same LGL cell lines also expressed high levels of lymphocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1). Engraftment of these LGL cell lines into severe combined immunodeficient (SCID) mice led to the development of leukemia and lymphomas. Examination of these SCID mice showed that their pathology was nearly identical to that observed in the original Tax-transgenic mouse model. Both the Tax-transgenic and engrafted SCID mouse models allow for the analysis of cellular events that are required for tumor development associated with HTLV infection and suggest that Tax expression may be responsible for the upregulation of certain cytokines and adhesion molecules that affect the infiltrating capabilities of HTLV-I-infected cells.     

In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis

Recombinant interferon beta (IFNbeta) benefits patients with relapsing remitting multiple sclerosis (MS), but the mechanisms of action are unknown. We studied in vivo immunologic effects of IFNbeta treatment and their relationship to clinical efficacy. Cytokines were measured in blood and CSF from MS patients participating in a placebo-controlled phase III clinical trial and an open-label phase IV [corrected] tolerability study of IFNbeta-1a. Additionally, immunologic studies were conducted in animals with proteolipid protein (PLP)-induced chronic relapsing experimental autoimmune encephalomyelitis. Single intramuscular (IM) injections of IFNbeta-1a (6 MIU, 30 microg) were associated with significant in vivo upregulation of interleukin-10 (IL-10) and IL-4 but not IFNgamma mRNA in peripheral blood mononuclear cells. Forty-eight hours after each IFNbeta-1a injection, serum IL-10 levels increased and remained elevated for 1 week. IFNbeta-1a recipients in the placebo-controlled phase III clinical trial showed significantly increased concentrations of CSF IL-10 after 2 years of treatment. This response correlated with a favorable therapeutic response. Exposure of PLP-reactive murine T-cell lines to IFNbeta resulted in increased antigen-driven expression of IL-4 and IL-10 and reduced encephalitogenicity. IFNbeta-1a injections induce systemic and intrathecal immunosuppressive cytokines. Myelin-specific T cells treated with IFNbeta-1a demonstrate increased immunosuppressive cytokine expression and reduced encephalitogenicity. The relationship between increased CSF IL-10 and response to therapy suggests that induction of IL-10 is a mechanism underlying IFNbeta-1a effects in MS patients.     

Apoptosis enhanced by soluble factor produced in feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV)-infected cells have been shown to undergo apoptosis by treatment with tumor necrosis factor alpha (TNF-alpha). This study detected a soluble factor which enhanced the apoptosis induced by TNF-alpha treatment. The sensitivity to TNF-alpha in the induction of apoptosis in a feline fibroblastoid cell line (CRFK) cells was significantly enhanced when the culture supernatant of FIV-infected CRFK cells or plasma samples from FIV-infected cats was added to the culture. These findings suggested that FIV infection induces production of a soluble factor which enhances CRFK cells sensitivity to TNF-alpha induction of apoptosis both in vitro and in vivo. This factor may contribute to the loss of lymphocytes in cats infected with FIV.     

Interleukin-12 enhances CD26 expression and dipeptidyl peptidase IV function on human activated lymphocytes

Research of a cellular pathway activated by IL-12 which may result in new therapeutical approaches for IL-12, led us to find an intriguing relationship between IL-12 and CD26/DPPIV ectopeptidase on activated T cells. Both the percentage and median fluorescence intensity (MFI) of CD26+ cells in the PHA-stimulated PBMC or lymphoblasts increased when IL-12 (optimum dose, 2 ng/ml) was present. Maximum CD26 expression was observed on day-2 cultures of lymphoblasts, the presence of IL-12 receptor probably being necessary for this upregulation. In addition, CD26 upregulation correlated with enhanced DPPIV function. Enzyme affinity and secretion of the soluble form of DPPIV were not affected by IL-12. Kinetic behaviours of Ag expression and enzymatic activity support a different CD26 regulation pathway by IL-12. These data suggest that the correlation found in vivo between the CD26 expression and Th1-like immune responses is due to this IL-12-dependent upregulation.     

Antiprogestin-mediated inactivation of cytochrome P450 3A4

Interleukin-12 (IL-12) is a heterodimeric cytokine that is central to the development of T helper 1-dependent cellular immunity. Although this cytokine has potential therapeutic application as an antineoplastic agent, the systemic infusion of IL-12 has led to toxic fatalities; hence, restriction of expression of IL-12 to the microenvironment of target tumor cells has obvious appeal. In this study, we examined whether tumor cells that were liposome-transfected with IL-12 could enhance the induction of cytolytic lymphocyte immunity to the native tumor. The plasmid expression vector that we used has several useful features including replication to high copy number as an episome and a polycistronic message enabling the production of both the p35 and p40 subunits of IL-12 without alternative splicing; up to 3 ng/mL/10(6)/48 hours of IL-12 was produced following transfection. Tumor cells transfected with IL-12 were superior to untransfected cells in the induction of lymphocyte-mediated cytolysis. IL-12 transfectants induced a heterogeneous population of natural killer, lymphokine activated killer, and cytolytic T lymphocytes, the latter of which exhibited tumor-specific activity. Our studies suggest that liposome-mediated transfection of tumor cells with an episomal, high copy number plasmid vector expressing both IL-12 subunits is a promising approach to cancer vaccination, a strategy that could be implemented ex vivo in treating malignancies such as metastatic ovarian cancer.     

IL-4-independent regulation of in vivo IL-9 expression

We focused on the role of IL-4 in the regulation of the Th2 cytokine IL-9. In vivo, IL-9 mRNA was detected in lymph nodes after immunization with soluble Ags. IL-9 expression preceded that of IL-4, and was not affected in IL-4 knockout mice. In contrast, a significant decrease of IL-9 message was observed in IL-10-deficient mice, indicating a role for this cytokine in the induction of IL-9 production. Treatment with anti-CD4 Ab and analysis of purified CD4 cells confirmed that IL-9 was produced by CD4+ cells. Moreover, similarly to what has been reported for IL-4, IL-9 message induction was strongly decreased by infection with lactate dehydrogenase-elevating virus. IL-9 mRNA was also detected after in vivo stimulation with anti-CD3 Ab. In this model, IL-9 expression followed that of IL-4, but was not reduced in IL-4-deficient mice. This contrasts with in vitro stimulation in which, as reported in humans, IL-9 expression in lymphocytes incubated with anti-CD3 Ab and costimulatory molecules appeared as a late event, and was partly dependent on IL-4. In vitro IL-9 secretion was reduced significantly by addition of anti-IL-4 Ab, as well as in lymphocytes from IL-4 gene-deficient mice. Taken together, our results indicate that the Th2 cytokine IL-9 can be expressed by both IL-4-dependent and -independent pathways.     

Incubation time for feline immunodeficiency virus cultures

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.