Generation of expression constructs that secrete bioactive alphaMSH and their use in the treatment of experimental autoimmune encephalomyelitis.

alpha Melanocyte-stimulating hormone (alphaMSH) is a 13 amino acid peptide with potent anti-inflammatory effects. We created two DNA expression constructs (miniPOMC and pACTH1-17) that encode bioactive versions of the alphaMSH peptide, and tested these constructs for therapeutic effects in experimental autoimmune encephalomyelitis (EAE). Each construct contained the sequences for alphaMSH, as well as the sequences that are involved in the secretion and processing of the POMC gene with the assumption that these sequences would promote processing and release of the encoded alphaMSH peptide. The differences between the two constructs lie at the C-terminal end where amino acids necessary for amidation of alphaMSH were included in only the pACTH1-17 construct. These two constructs were tested in vitro in bioassays, and in vivo in a mouse model of EAE. The results show that although bioactive peptides are secreted from cells transfected with either construct, there appears to be a significant therapeutic effect only with the pACTH1-17 construct which contains the extra C-terminal amino acids. The data suggest that it is possible to engineer DNA expression vectors encoding small secreted peptides such as alphaMSH, and that similar type constructs may be useful as therapeutics for the treatment of inflammatory diseases.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells. Transduction efficiencies of CD34+ cells ranged between 22 and 27%. Differentiation of CD34+ cells into T cells or monocytes was not significantly altered by the transduction process. HIV-1 replication in monocytes and CD4+ T cells derived from CD34+ cells transduced with the intracellular antibody gene was significantly reduced in comparison with the degree of HIV replication seen in monocytes and CD4+ T cells derived from CD34+ cells transduced with the neomycin resistance gene alone. Further, T cells and monocytes derived from CD34+ cells transduced with the intracellular antibody gene were demonstrated to express the sFvtat1-Ckappa transgene by RT-PCR and had a selective growth advantage in cultures that had been challenged with HIV-1. These data demonstrate that sFvtat1-Ckappa inhibits HIV-1 replication in T cells and monocytes developing from CD34+ cells and supports the continuing development of a stem cell gene therapy for the treatment of HIV-1 infection.

Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies.

We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells from HIV-infected individuals that express intracellular antibodies against HIV-1 gp120 or Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.

Cyclic AMP-mediated growth arrest is associated with increased expression of human T cell leukemia virus type I structural and transforming genes.

The effect of increased intracellular cyclic AMP levels on gene expression of the human T cell leukemia virus type I (HTLV-I) provirus was examined. Induction of infected cells to produce elevated levels of cyclic AMP was associated with specific increases in viral surface antigen expression, protein synthesis, p24 release into the supernatant, and RNA levels. The patterns of HTLV-I proviral gene expression observed support results from transfection experiments regarding the function of Tax, Rex, and cyclic AMP in HTLV-I gene regulation. As evidenced by thymidine incorporation, treatment of the infected cells to produce cyclic AMP caused reversible growth arrest. The data indicate that HTLV-I RNA and protein synthesis can proceed at an elevated level in the absence of cell growth. Sustained increases in the intracellular level of cyclic AMP may represent a method for enriching cell cultures in HTLV-I proteins.

Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture.

Human T-cell leukemia virus type 1 (HTLV-1) immortalizes human CD4+ T lymphocytes in culture. Previous studies show that in the context of a herpesvirus saimiri vector, the sequence of the X region at the 3' end of the HTLV-1 genome is also capable of immortalizing CD4+ lymphocytes in the absence of HTLV-1 structural proteins. The X region of HTLV-1 encodes two trans-acting viral proteins, the 42-kDa Tax protein and the 27-kDa Rex protein. Infection of human cord blood cells with herpesvirus saimiri recombinants which contain HTLV-1 X region sequences defective for expression of tax, rex, or both tax and rex demonstrates that tax function is necessary and sufficient for immortalization of primary human CD4+ cord blood lymphocytes in culture in the context of the herpesvirus saimiri vector.

Impact of 90Sr on mouse natural killer cells and their regulation by alpha interferon and interleukin 2.

Male CBA/SU mice were exposed to ionizing radiation by intraperitoneal injection of the bone-seeking beta-emitter 90Sr. NK-cell lytic activities in spleen, peripheral blood, and lymph nodes were severely depressed or completely abolished. In contrast, production of the NK regulatory proteins alpha interferon (IFN-alpha) and interleukin 2 (IL-2) was normal 5-8 weeks after 90Sr injection. IFN-alpha, produced in vivo or in vitro by cells from injected mice, was able to enhance strongly NK lytic activities. These data indicate that 90Sr acts on the bone marrow, where it interferes with the maturation and seeding of NK precursor cells. The mechanisms regulating NK activities in peripheral organs remained relatively unchanged. Finally, we did not detect any major organ redistribution of NK cells as a result of 90Sr irradiation.

Mutant monoclonal antibodies with select alteration in complement activation ability. Impact on immune complex functions in vivo.

Mutagenesis of mAb is a useful means for studying the biologic and pathologic functions of immune complexes. Treatment of the Hy-1.2 hybridoma-producing IgG2a-anti-TNP antibodies with ethylmethanesulfonate provided us with a mutant clone, producing antibodies with reduced capacity for C activation. The antibodies retained normal Ag-binding capacity, staphylococcal protein A reactivity, and association to FcR for IgG on murine macrophages. No significant polypeptide deletion or class-switch was observed, but a significant change in clonotype was revealed by IEF. Intravenous injection of the mutant antibodies in immune complex form induced different tissue distributions of Ag in mice; i.e., more in kidneys and less in spleen, and developed more mesangial deposits in renal glomeruli compared with those of the wild type. Moreover, the production of granulomatous lesions in vivo caused by immune complexes of TNP-Sepharose was augmented by using mutant antibodies. These lesions demonstrated an enhanced accumulation of macrophages with multinucleated giant cells. Availability of this kind of mutant mAb is thus helpful in the elucidation of the biologic functions and consequences of immune complexes.

In human monocytes a strong correlation exists between expression of the M3 antigen, Fc-mediated phagocytic activity and failure to participate in extracellular antibody-dependent cytotoxicity.

Human fresh blood monocytes can phagocytize and lyse antibody-coated target cells by contact with membrane Fc receptors. Recently, the monocyte differentiation antigen Leu M3 has been described to be associated with monocyte/macrophage maturation pathway and to be linked to functionally distinct monocyte subsets. In the present study peripheral blood monocytes were separated into M3+ and M3- subsets, and evaluated for their ability to mediate antibody-driven effector functions. A clear cut functional difference could be demonstrated. M3+ monocytes phagocytize antibody-coated sheep red cells but do not carry out contact-mediated extracellular lysis. In contrast, M3- monocytes have exactly the opposite functional features and they mediate cytolysis without exhibiting any phagocytic activity. By using phenotypically defined clones of the U937 histiocytic cell line, the segregation of different lytic abilities and their linkage of the M3 phenotype have been confirmed.

Inhibition of natural killer cell cytotoxicity by a monoclonal antibody directed against adhesion-mediating protein gp 90 (CD18).

Contact is required between the effector natural killer (NK) or cytotoxic killer T cell and its corresponding target in order for efficient lysis to occur. Several surface molecules of different types are involved in this process. Here we could show that Fab fragments from a murine monoclonal antibody reacting with gp 90, the human leucocyte common antigen CD18, are extremely efficient in blocking human NK of killer T cells, regardless of whether the target has or does not have the antigen. In contrast, no impact of the antibody was observed when the effector cells were of murine origin, again regardless of whether the target cell did or did not display the leucocyte common antigen. The inhibition could be shown to occur at the level of blockage of target-conjugate formation. This means that the functional display of effector/target gp 90 on the effector but not the target cell is necessary for efficient lysis to occur both in NK and killer T cell systems.

A specific assay measuring binding of 125I-Gp 120 from HIV to T4+/CD4+ cells.

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4+ (T4+) cells. 125I-Gp120 could be shown to selectively bind to CD4+ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4+ cells. Monoclonal antibodies to other cell surface components do not interfere with 125I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block 125I-Gp120 binding, though with variable titers. We believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding.

Lipoxin A-induced inhibition of human natural killer cell cytotoxicity: studies on stereospecificity of inhibition and mode of action.

Human leukocyte-derived lipoxin A (LXA; 5S,-6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) inhibits the cytotoxic activity of human natural killer (NK) cells. LXA and three of its isomers were prepared by total organic synthesis and assayed for activity with human NK cells. Dose-response studies showed that biologically derived LXA and synthetic LXA were equally effective in inhibiting NK cell cytotoxicity. 6S-LXA, with its 6S-OH group in an (S) configuration, proved to be approximately half as potent as LXA. In contrast, 6S-11-trans-LXA and 11-trans-LXA displayed virtually no inhibitory activities. The methyl esters of both LXA and 6S-LXA proved to be more potent than their corresponding free acids. Thus, LXA inhibition of NK cells displays clear-cut stereochemistry. In the absence of putative inhibitors, NK cells bind to their targets to form conjugates. This event is followed by polarization of the NK Golgi apparatus, which moves towards the plasma membrane that is in contact with the target cell. However, in the presence of either the methyl ester or free acid of LXA, the Golgi apparati of NK cells bound to their targets were randomly oriented. In contrast, neither 6S-11-trans-LXA nor the potent NK inhibitor prostaglandin E2 affected the polarization. Furthermore, although prostaglandin E2 resulted in a decrease in NK-target cell binding efficiency, LXA and its isomers failed to affect conjugate formation. Together these results indicate that LXA-induced inhibition of NK cytotoxicity does not act on NK cell binding but may block cytotoxicity by disrupting "signals" involved in the specific orientation of the Golgi. Thus, this latter event may appear to be important in cytotoxicity.

NK-cell stimulating properties of a membrane proteoglycane from non-capsulated Klebsiella pneumoniae biotype a.

Upon testing the individual fractions of a composite bacterial vaccine for biological activities, a potent immuno-stimulatory capacity could be demonstrated within a crude membrane proteoglycan preparation from Klebsiella pneumoniae. One of its characteristic features was the capacity to induce alpha-type interferon and increased NK activity in vivo in mice, following intraperitoneal or oral administration. A highly purified fraction from the crude preparation was obtained using alkaline hydrolysis, delipidation and size fractionation. This fraction was shown to be a very potent inducer of NK cells in vivo or in vitro, where in the latter systems concentrations as low as 0.1 microgramme per ml were highly efficient.

Action of novel eicosanoids lipoxin A and B on human natural killer cell cytotoxicity: effects on intracellular cAMP and target cell binding.

Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells.

Human peripheral blood null lymphocytes stimulated by Staphylococcus aureus Cowan I produce atypical acid-labile interferon in vitro.

Peripheral blood mononuclear leucocytes (PBLs) stimulated in vitro by heat-killed formaldehyde-fixed Staphylococcus aureus Cowan I (SACoI) produced acid-labile alpha interferon (IFN-alpha) and, to various extents, also IFN-gamma. The IFN producers resided in nylon wool-nonadherent cells, and monocytes suppressed SACoI-induced IFN responses. Further separation of nonadherent PBLs in accordance with expression of surface antigenic markers was performed with a 'panning' technique. The SACoI induced production of IFN in cells that carried neither surface immunoglobulins nor OKT3-defined antigens. These cells were also characterized as OKM1- and OKT10-negative. In contrast, cells with natural killer (NK) activity against K562 erythroleukaemia cells were located in both OKM1- and OKT10-positive and -negative cells. At centrifugation on Percoll density gradients, cells with NK activity and IFN response against SACoI were recovered from light gradient fractions that contained mainly large granular lymphocytes (LGL). Furthermore, the IFN producers were enriched by removal of sheep erythrocyte-rosetting T cells from the Percoll fractions. These SACoI-induced IFN-producing PBLs are LGL but lack certain antigens that are frequently found on NK cells.

Inhibition of human natural killer cell activity by (14R,15S)-14,15-dihydroxy-5Z,8Z,10E,12E- icosatetraenoic acid.

The interactions between products of the 15-lipoxygenase cascade and human natural killer (NK) cell activity have been studied. Addition of human leukocyte-derived (14R,15S)-14,15-dihydroxy-5Z,8Z,10E,12E-ic osatetraenoic acid (14,15-DiHETE) to the NK cytotoxicity assay against K562 target cells resulted in inhibition of NK cell activity, whereas addition of other 15-lipoxygenase-associated metabolites [i.e., (15S)-15-hydroperoxy-5Z,8Z,11Z,13E-icosatetra eno ic acid, (15S)-15-hydroxy-5Z,8Z,11Z,13E-icosatetraenoic acid, and (8R,15S)- and (8S,15S)-8,15-dihydroxy-5Z,9E,-11E,13E-icosat etr aenoic acid isomers] resulted in little or no inhibition of NK function. Dose-response studies indicate that leukocyte-derived 14,15-DiHETE and 14,15-DiHETE methyl ester, at micromolar concentrations, inhibit NK function even in the presence of 2.5% fetal calf serum. Synthetic 14,15-DiHETE prepared by total organic synthesis displayed similar biological activities over identical dose ranges. These icosanoids do not inhibit NK target cell binding and they exert only a variable effect in either antibody-dependent cytotoxicity or cytotoxic T-lymphocyte assays. These results demonstrate that the 14,15-DiHETE inhibits NK cell function in vitro. Moreover, they suggest that activation of the 15-lipoxygenase cascade and formation of 14,15-DiHETE in vivo may provide a mode of immune regulation.

Free oxygen radicals are not detectable by chemiluminescence during human natural killer cell cytotoxicity.

Mononuclear cells isolated from peripheral blood of normal donors produce free oxygen radicals (FR), detectable by chemiluminescence (CL), when interacting with target cells during natural killer (NK) cell lysis. FR-producing cells were found to have monocyte characteristics and gave a positive CL reaction when mixed at low concentration (0.5%) with purified NK cells. No correlation was found between susceptibility to NK cell lysis and capacity to induce CL with different target cell lines. Using high and low molecular FR scavengers, no NK cell inhibition was seen with superoxide dismutase, cytochrome c, and catalase, whereas some inhibition was seen with 4,5-dihydroxy-m- benzenedisulphonic acid (Tiron) and 2,3-dihydroxybenzoate. These compounds, however, required higher concentrations than used for inhibition of CL, suggesting an alternative action of these compounds. Normal levels of NK cell activity were found in two patients with chronic granulomatous disease, who were genetically incapable of producing detectable amounts of FR. As a result, it is concluded that human NK cells do not produce large amounts of FR during killing and that FR are unlikely to be the lytic end product. Nevertheless, neither a low degree of FR formation in NK cells nor a more subtle signal-transmitting role of FR during NK cell triggering can be excluded.

Natural killer activity in (C57BL/6 X DBA/2)F1 hybrids undergoing acute and chronic graft-vs.-host reaction.

The present findings demonstrate that a total i.v. transfer of 100 X 10(6) C57BL/6 (B6) parental spleen cells into untreated (C57BL/6 X DBA/2)F1 hybrids (B6D2F1) resulted in acute runting, which was associated with a significantly elevated graft-vs.-host (GVH) index over a one-month period following GVH induction. Furthermore, this B6-induced acute GVH disease was associated with a marked depression of natural killer (NK) cell activity (spleen and peripheral blood) (with or without addition of mouse fibroblast interferon), which correlated with lymphoid cell hypocellularity, prominent splenic extramedullary hematopoiesis (EMH), and parallel depressions of both concanavalin A- and lipopolysaccharide-induced mitogenesis. Significantly increased killing by antibody-dependent cellular cytotoxicity of antibody-coated chicken red blood cells, as well as increased T cell killing of the NK-insensitive cell line P815 (as compared to the significantly decreased killing of the NK-sensitive cell line YAC-1) was also observed in the spleens of this 100 X 10(6) B6-injected F1 group. In marked contrast to this 100 X 10(6) B6-injected acute GVH group, untreated mice injected i.v. with the same or greater numbers of parental DBA/2 spleen cells (100 X 10(6)-150 X 10(6) DBA/2 spleen cells) exhibited a milder and more chronic form of GVH disease, which was not associated with a significant decrease of NK activity. It was of considerable interest that a total i.v. transfer of 50 X 10(6) B6 spleen cells (i.e. one-half of that required to produce acute GVH, markedly depressed NK, and prominent splenic EMH) into B6D2F1 hybrids also resulted in a more chronic form of GVH disease, but was associated with significantly increased levels of NK activity at two weeks post GVH induction.