Gene therapy for lung disease.

Gene therapy is a new field of medical research that has great potential to influence the course of treatment of human disease. The lung has been a particularly attractive target organ for gene therapy due to its accessibility and the identification of genetic deficits for a number of lung diseases. Several clinical trials have shown evidence of low levels of gene transfer and expression, but without any benefit to the patients involved. Thus, current studies are focusing on further research and technological improvements to the vectors. Gene therapy is now beginning to benefit from a shift in emphasis from clinical trials to the development of better tools and procedures to deliver gene therapy to the bedside.

Nonspecific inflammation inhibits adenovirus-mediated pulmonary gene transfer and expression independent of specific acquired immune responses.

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (beta-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1alpha, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1alpha, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-alpha, IL-6, IL-1beta, and IFN-gamma were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung.

In vivo delivery of interleukin-4 by a recombinant vaccinia virus prevents tumor development in mice.

To study the immunotherapeutic potential of interleukin-4 (IL-4) delivered in vivo via a recombinant vaccinia virus, a thymidine kinase-negative (TK-) vaccinia virus that expressed the murine IL-4 gene (VV1/IL-4) was constructed. When mice were inoculated with 10(7) plaque-forming units (pfu) of VV1/IL-4 subcutaneously (s.c.), 10(5) pfu/cm2 were found in skin, and smaller numbers in liver and kidney between 1 and 7 days after infection; few viral pfu were found in spleen and lung, or in any organ after intravenous infection. This suggested that recombinant vaccinia viruses might be most efficient at delivery of cytokine genes to the skin. Because IL-4 has recently been found to have potent anti-tumor activity, the effect of recombinant virus infection on the development of s.c. tumors was studied. A single s.c. inoculation with VV1/IL-4 delayed the development of NCTC 2472 tumors, but when VV1/IL-4 was inoculated s.c. weekly for 8 weeks, tumor development was completely prevented in 93% of mice. Similarly, the development of M-3 melanoma tumors was also prevented by weekly s.c. inoculations of VV1/IL-4. About 40% of mice treated with control VV2/beta gal by the same regimen also failed to develop tumors. Weekly virus treatment did not prevent NCTC 2472 tumor development in athymic nu/nu mice, suggesting that mature T cells are required for expression of VV1/IL-4 induced antitumor activity. Thus, recombinant vaccinia viruses may be especially well suited for convenient therapeutic delivery of immunomodulator genes to skin-related sites.

The genomic structure of the murine ICSBP gene reveals the presence of the gamma interferon-responsive element, to which an ISGF3 alpha subunit (or similar) molecule binds.

ICSBP, a member of the interferon regulatory factor family, is expressed predominantly in lymphoid tissues and is induced by gamma interferon (IFN-gamma). We have studied the genomic organization of the murine ICSBP gene and its 5' upstream region. The murine ICSBP gene (Icsbp) is present as a single copy on chromosome 8 and consists of nine exons. Transcription initiates at two juxtaposed sites downstream from the TATA and CAAT boxes and produces two species of ICSBP mRNA (3.0 and 1.7 kb), presumably by differential usage of poly(A)+ signals. A sequence from -175 to -155 was identified to be an IFN response region that conferred IFN-gamma induction upon a heterologous promoter in lymphoid cell line EL4. This region includes a motif, TTCNNGGAA, designated the palindromic IFN response element (pIRE), to which an IFN-gamma-inducible, cycloheximide-sensitive factor(s) binds. A similar palindromic motif was found in the upstream region of the murine IRF-1 gene, the IFN-gamma activation site of the guanylate-binding protein gene and the IFN-gamma-responsive region of the Fc receptor type I gene, all of which competed with the pIRE for factor binding in gel mobility shift assays. We show that the pIRE binding factor reacts with the antibody against the 91-kDa subunit of ISGF3 alpha recently shown to bind to the IFN-gamma activation site. These results suggest that this factor is related to the IFN-gamma activation factor and contains the 91-kDa subunit of ISGF3 alpha. Taken together, pIRE represents an IRE that is distinct from the classical IFN-stimulated response element and that is capable of conferring IFN-gamma induction through the binding of the 91-kDa ISGF3 alpha subunit (or an antigenically similar molecule).

Activation of immunoglobulin control elements in transgenic mice.

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EmukCAT, contains the Ig heavy chain enhancer (Emu) and the kappa light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, delta EmukCAT, CAT is under the control of the kappa promoter alone. Emu and kappa relative activity were assessed by CAT assay. In EmukCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In delta EmukCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon-gamma (IFN-gamma) increased CAT expression to varying extents in cells derived from EmukCAT mice but not in spleen cells prepared from delta EmukCAT mice. Thus, the presence of Emu, in addition to the kappa promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EmukCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN-gamma caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.

Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.

An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes.

Interferons (IFNs) induce transcription of major histocompatibility complex (MHC) class I genes through the conserved IFN consensus sequence (ICS) that contains an IFN response motif shared by many IFN-regulated genes. By screening mouse lambda ZAP expression libraries with the ICS as a probe, we isolated a cDNA clone encoding a protein that binds the ICS, designated ICSBP. Protein blot analysis with labeled oligonucleotide probes showed that ICSBP binds not only the MHC class I ICS but also IFN response motifs of many IFN-regulated genes, as well as a virus-inducible element of the IFN-beta gene. The ICSBP cDNA encodes 424 amino acids and a long 3' untranslated sequence. The N-terminal 115 amino acids correspond to a putative DNA-binding domain and show significant sequence similarity with other cloned IFN response factors (IRF-1 and IRF-2). Because of the structural similarity and shared binding specificity, we conclude that ICSBP is a third member of the IRF gene family, presumably playing a role in IFN- and virus-mediated regulation of many genes. Although IRF-1 and IRF-2 share some similarity in their C-terminal regions, ICSBP shows no similarity to IRF-1 or IRF-2 in this region, suggesting that it is more distantly related. We show that ICSBP mRNA is expressed predominantly in lymphoid tissues and is inducible preferentially by IFN-gamma. The induction by IFN-gamma appears to be predominant in lymphocytes and macrophages, implying that ICSBP plays a regulatory role in cells of the immune system. The presence of multiple factors that bind common IFN response motifs may partly account for the complexity and diversity of IFN action as well as IFN-regulated gene expression.

Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide.

The clinical manifestations of AIDS (acquired immune deficiency syndrome) often include neuropsychiatric and neurological deficits, including early memory loss and progressive dementia. HIV (human immunodeficiency virus), the aetiological agent of AIDS, is probably carried by infected macrophages in the central nervous system. The virus enters cells by binding its envelope glycoprotein gp120 to the CD4 antigen present on brain and immune cells. From the data reported in this paper, we now suggest that the neuronal deficits associated with HIV may not be entirely a result of infectivity, but that gp120 shed from HIV could directly produce the neuropathology as a result of its interference with endogenous neurotrophic substances. It is known that an analogue of a sequence contained in vasoactive intestinal peptide (VIP) occurs in all known sequenced gp120 isolates and that VIP is important for neuronal survival in cell culture. Here we show that purified gp120 from two diverse HIV isolates and a recombinant gp120 from a third isolate were all potent in specifically producing significant neuronal cell death in dissociated hippocampal cultures derived from fetal mice, and that this could be reduced by monoclonal antibodies against the murine CD4 antigen and completely antagonized by VIP.

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I. BCGF II-like activity of human BCGF.

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.

Enhancement of non-Candida antibody responses by Candida albicans cell wall glycoprotein.

Two cell wall glycoprotein extracts from Candida albicans (glycoprotein [GP] and peptidoglucomannan [PGM]) were tested for their influence on antibody responses to type III pneumococcal polysaccharide and sheep erythrocytes. GP was isolated from lipid-extracted cell walls with ethylenediamine, whereas PGM was extracted with dilute sodium hydroxide. Both glycoproteins increased the number of antibody-producing plaque-forming cells in the spleens of mice immunized with type III polysaccharide or sheep erythrocytes, although PGM appeared to be about 10 times more effective. PGM could be administered up to 3 days prior to immunization with sheep erythrocytes to elicit enhancement; it did not have to be administered by the same route as the immunogen to cause significant enhancement. Enhancement did not appear to be the result of a direct mitogenic effect of GP and PGM on lymphocytes, nor did these glycoproteins appear to stimulate the production of B-cell growth factors or interleukin 2.

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation.

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.

Functional analysis of the immunosenescence of the human B cell system: dissociation of normal activation and proliferation from impaired terminal differentiation into IgM immunoglobulin-secreting cells.

The abilities of B cells from 24 young (mean 26 yr) and 24 elderly (mean 86 yr) humans to proliferate and differentiate into immunoglobulin-secreting cells (ISC) were investigated. Initial studies in young subjects demonstrated that a Staph protein A (SpA)-driven system could simultaneously assess the proliferative and differentiative capabilities of B cells resulting in IgM production. B cell proliferative responses were found to be partially T cell-dependent, whereas differentiation was absolutely T cell-dependent. Also, no significant differences could be detected in the abilities of nonproliferating allogeneic and autologous T cells to support B cell responsiveness. Although B cells from elderly subjects continuously exposed to SpA displayed proliferative responses equal to young subjects, the differentiation of B cells from elderly subjects into IgM ISC was markedly reduced as compared to young subjects. Analyses of results from co-culture experiments showed that the differentiation impairments of B cells from some elderly subjects could be partially corrected by allogeneic T cells from young subjects, whereas the impairments of others were more refractory. Moreover, T cells from elderly subjects were able to promote the differentiation of B cells from young subjects. Other experiments in elderly subjects showed that significant impairments of B and T cell functions rarely coexisted and that compensatory increases in B or T cell function were not evident. Thus, B cells from certain elderly humans have intrinsic impairments of differentiation required for optimal IgM production even though activation and proliferation remain normal in the presence of SpA. These impairments in differentiation are sometimes improved by T cells from young subjects, although in some elderly individuals, the differentiative impairments fail to be reversed.

Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.

A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.