Intracisternal rSV40 administration provides effective pan-CNS transgene expression.

Potential genetic treatments for many generalized central nervous system (CNS) diseases require transgene expression throughout the CNS. Using oxidant stress and apoptosis caused by HIV-1 envelope gp120 as a model, we studied pan-CNS neuroprotective gene delivery into the cisterna magna (CM). Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase or glutathione peroxidase were injected into rat CMs following intraperitoneal administration of mannitol. Sustained transgene expression was seen in neurons throughout the CNS. On challenge, 8 weeks later with gp120 injected into the caudate putamen, significant neuroprotection was documented. Thus, intracisternal administration of antioxidant-carrying rSV40 vectors may be useful in treating widespread CNS diseases such as HIV-1-associated neurocognitive disorders characterized by oxidative stress.

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe.

Gene transfer to central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce human neurons and microglia effectively in vitro and in rodent brains in vivo, so we tested rSV40s gene transfer to rhesus monkey CNS in vivo, to characterize the distribution, duration and safety of such gene delivery. We used rSV40s carrying HIV-1 RevM10 with a carboxyl-terminal AU1 epitope tag as a marker, and others with the antioxidant enzymes, Cu/Zn superoxide dismutase and glutathione peroxidase. Vectors were injected stereotaxically into the caudate nucleus. Transgene expression was studied at 1 and 6 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, with a longitudinal extent of 20 mm. In neurons and, more rarely, microglial cells, transgene expression remained strong throughout the 6-month study period. Astrocytes and oligodendroglia were not transduced. No evidence of inflammation or tissue damage was observed. SV40-derived vectors may thus be useful for long-term gene expression in the monkey brain and, potentially, in the human brain.

In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors.

In cystic fibrosis (CF), respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress toward gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF, we examined the feasibility of using recombinant SV40-derived vectors (rSV40s), which may circumvent some of these obstacles. To accommodate the large cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We, therefore, tested whether 'gutless' rSV40s could be packaged and were able to express a functional human CFTR cDNA. The results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein, which localized to the plasma membrane and restored channel function to CFTR-deficient cells. Similarly, in vivo experiments delivering rSV40-CFTR to the lungs of Cftr-/- mice resulted in a reduction of the pathology associated with intra-tracheal P. aeruginosa challenge. rSV40-CFTR-treated mice had less weight loss when compared with control-treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the broncho-alveolar lavage. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted.

Clonal depletion in neonatal tolerance.

Specific unresponsiveness can be induced in neonatal and adult BALB/c mice by antibody against antigen-specific receptor (antireceptor antibody). When heterologous antireceptor antibody is used in the indirect fluorescence technique, the number of fluorescent cells in these animals is significantly lower than in normal animals. Fluorescent cells appear after a relatively brief incubation of cells from adult-suppressed animals, whereas no fluorescent cells are detected when cells from neonatally treated animals are incubated briefly. Evidently, treating neonatal mice with antireceptor antibody specifically depletes the antigen-responsive clone. In contrast, antireceptor antibody causes reversible blockade of responsive cells in adult-suppressed animals.

Neonatal tolerance induced by antibody against antigen-specific receptor.

Specific immunologic unresponsiveness is induced by injecting adult or neonatal mice with antibody against antigen-specific receptor (antireceptor antibody). Suppression in mice treated as adults lasts several weeks, and cells from these suppressed mice respond normally in culture. In contrast, unresponsiveness induced in neonatal mice is long-lasting; cells from these mice do not respond in culture and do not affect the response of normal cells. Evidently, antireceptor antibody reversibly blocks antigen receptors in adult animals, but induces unresponsiveness in neonatal mice by depleting the clone of receptor-bearing cells.

Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth.

Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.

Immunohistology of malignant rabbit fibroma virus--a comparative study with rabbit myxoma virus.

Malignant rabbit fibroma virus (MV) causes a syndrome that consists of disseminated malignant tumors and immunosuppression complicated by severe Pasteurella multocida infection and death. Tissues from rabbits given MV and rabbit myxoma virus were examined by direct immunofluorescence with the use of antibody against virus antigens. Primary and metastatic tumors caused by MV and rabbit myxoma virus were composed of soft tissue cells containing virus antigens. Skin appendages and epidermis overlying the respective tumors showed scant MV but abundant myxoma virus antigen. Both viruses were present systemically in the reticuloendothelial system. Epithelial cells from the liver, kidney, and lung of myxoma virus-infected rabbits contained virus, whereas in MV tumor-bearing rabbits, these cells were uninvolved. However, nasal mucosal and conjunctival epithelia, the locations of Pasteurella infection, showed squamous metaplasia and contained large amounts of MV and myxoma antigens. By analogy to other respiratory tract pathogens, these epithelial changes were probably etiologically significant for development of pasteurellosis in rabbits bearing virus-induced tumors. Thus by immunopathologic as well as clinical examination, MV produces a syndrome distinct from that seen with rabbit myxoma virus. MV induced severe immunosuppression despite T-lymphocyte hyperplasia in the lymphoid tissues observed. The combination of a systemic virus infection, epithelial alterations that impaired clearance mechanisms, and immunologic dysfunction is likely to contribute to the inability of rabbits given MV to survive their gram-negative infection.

Malignant rabbit fibroma virus: observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor.

The clinical, histopathologic, and cultural characteristics of a newly isolated poxvirus, malignant rabbit fibroma virus (MV), were investigated. MV was isolated from tumors induced by an uncloned stock of Shope fibroma virus (SFV). MV, SFV, and rabbit myxoma virus were compared. Similarly to myxoma virus, MV grew to higher titer in vitro than did SFV and produced plaques rather than foci on rabbit kidney cell monolayers. Unlike the local, self-limited fibroblastic proliferations observed in SFV recipients, MV and myxoma caused a fulminant clinical syndrome characterized by malignant histology, metastases, and supervening fatal gram-negative infection with Pasteurella multocida. MV induced a large, protuberant local tumor and discrete metastases histologically resembling myxosarcomas. Draining lymph nodes contained metastases and showed diffuse cortical hyperplasia. Kupffer's cells were prominent in the liver, and macrophages were abundant in the splenic sinusoids. The lungs and trachea were spared, but the conjunctiva and nasal mucosa showed squamous metaplasia and atypia, with overlying Pasteurella infection and underlying tumor. Myxoma virus infection produced similar mucosal changes, but both of these as well as the epidermis overlying the myxomas showed cytoplasmic virus inclusions. Neither the skin nor the epithelial surfaces overlying MV-induced tumors nor the tumors themselves contained virus inclusions. Thus the tumor syndrome caused by MV differed from other known rabbit tumors. Endonuclease restriction digests showed that the MV genome resembled, but was distinct from, rabbit myxoma virus. Opportunistic infection associated with MV-induced disseminated tumor may be an experimental model for the infectious complications that often supervene in host-tumor relationships.

The upstream region of the SP-B gene: intrinsic promoter activity and glucocorticoid responsiveness related to a new DNA-binding protein.

We identified and cloned the rabbit SP-B gene, encoding the pulmonary surfactant-associated protein, and sequenced its upstream region from -2635 to +428, including a much larger fragment of the upstream region than has previously been reported for an SP-B for any species. Rabbit SP-B showed substantial homology to its human counterpart in the coding and noncoding regions immediately upstream from the TATAA box. Using a luciferase (Luc) reporter gene (luc) construct we measured promoter activity with a 212-bp fragment (SPB212) from nucleotides (nt) -41 to -252, inclusive. SPB212 functioned as an active promoter in this assay. Further, we identified, cloned and sequenced the cDNA encoding a unique DNA-binding protein, N, that bound SPB212 at approx. -195. When the N cDNA was cloned into the expression vector pKC4 and cotransfected with the luc reporter construct, N significantly enhanced Luc production, but only in the presence of dexamethasone. Therefore, we identified and sequenced a functional promoter region upstream from rabbit SP-B, and isolated and characterized a DNA-binding protein that confers enhanced glucocorticoid responsiveness on this promoter.

Effect of virus infection on levels of transcripts for cellular genes.

Malignant rabbit fibroma virus (MV) induces tumors composed of proliferating cells, principally fibroblasts, and vasculature. These tumors are associated with large amounts of collagen and other connective tissue proteins. We studied the effect of MV infection on levels of mRNA for alpha 1 chains of collagens type I, III and V in RK-13 fibroblasts and alpha 2 chain of collagen type I. MV infection induces expression of specific collagen genes at particular time points after infection in vitro. Expression of these collagen genes is clearly different in MV-infected cells compared to uninfected cells. Transcript levels for a cellular transcription factor that regulates expression of alpha 1 chain type I collagen, cbf-a, were increased in MV-infected cells prior to the increase in type I collagen mRNA. The virus infection also specifically induced increased levels of mRNA for the cellular transcription factors c-fos and SP1. MV infection is therefore associated with increased levels of specific cellular mRNAs, and is correspondingly associated with increased mRNA for transcription factors that may regulate transcription of these genes. The ability of malignant fibroma virus to influence expression of cellular genes may be exerted through the cells' own transcription regulatory apparatus.

Sequence and analysis of the BamHI "D" fragment of Shope fibroma virus: comparison with similar regions of related poxviruses.

Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.

Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies.

The reactivity of two rat monoclonal antibodies was studied. These antibodies, A2R and A2C, bind a 32 kDa alveolar type II cell membrane receptor for surfactant protein A. A2R and A2C also bind apical cell membranes of ciliated and nonciliated cells of the conducting airways. Because this reactivity suggested possible utility in targeting those cells for therapeutic gene transfer, the binding activity of these two antibodies was examined in human tissues. In conducting airways, A2R and A2C bound apical epithelial cell membranes throughout the embryologic period studied: from 15 weeks of gestation, through maturity. Reactivity was more restricted to ciliated cells of the airways as maturation progressed. In the peripheral lung, A2C and A2R only bound most cells in the early developing lung, but mainly type II cells in mature lungs. Other normal tissues recognized by these antibodies included crypt lining cells of the adult and fetal stomach, large bile duct epithelium, and pancreatic acinar cells. All of these cells derive from embryonic foregut endoderm. Other normal tissues, both of endodermal and nonendodermal origin, were negative. Pulmonary carcinomas were studied. A2C and A2R recognized all non-small cell carcinomas of the lung tested. In contrast, none of the small cell carcinomas or carcinoid tumors of the lung were recognized by these antibodies. The function of p32 in these diverse cell types is not clear, but whatever its role in these tissues, antibodies versus p32 may potentially be used to target gene or drug therapy to the normal or malignant cells they recognize.

Dysplasia of the kidneys, liver, and pancreas: report of a variant of Ivemark's syndrome.

A newborn girl with respiratory distress due to bilateral pneumothorax was found to be anuric, and died at 18 hours of age. Autopsy revealed a large pancreatic cyst, multiple large hepatic cysts, congenital hepatic fibrosis, bilateral dysplastic kidneys, and dysplasia of the pancreas. These findings constitute a variant of Ivemark's syndrome of dysplasia of the pancreas, liver, and kidneys.

The histologic spectrum of the cutaneous mycobacterioses.

The authors examined the histopathology of cutaneous involvement in 31 cases of nonleprous mycobacterial infection. Cases include three patients with Mycobacterium kansasii infection, two with M. fortuitum infections, and one each with M. marinum and M. chelonei infections, as well as 18 with M. tuberculosis infections. In the remainder, species were not identified. The histopathologic picture was variable and often did not suggest mycobacterosis. The authors identified seven basic pathologic patterns of skin involvement: 1) abscess, 2) well-formed (tuberculoid) granulomas, 3) diffuse histiocytic infiltration, 4) panniculitis, 5) nonspecific chronic inflammation, 6) naked (sarcoidal) granulomas, and 7) rheumatoid-like nodules. Intermediate forms were also found. Some cases showed adnexal or epidermal involvement, while others showed variably distributed dermal infiltration. The results indicate that a wide variety of cutaneous, clinical, and histologic guises may be assumed by mycobacterial infections in normal and immunocompromised hosts.

SV40-based gene therapy vectors: turning an adversary into a friend.

For gene delivery to be of use, a situation suitable for delivery of genetic material, a specific genetic construct to be delivered and the appropriate means to deliver it are required. Simian virus-40 (SV40) gene therapy vectors for gene transfer may be an important advance in the latter category. While other vectors are variably limited for example by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc., recombinant viral vectors based on SV40 (rSV40) should not be similarly constrained. They are easily manipulated and produced at very high titer, stable, apparently lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in almost any cell type, whether resting or dividing. The major limitation of SV40-derived vectors is packaging capacity, which restricts insert sizes. The rationale for developing SV40 as a gene therapy vector is reviewed, based on what is known of wild-type SV40. Studies with rSV40 gene transfer have focused mostly on hematopoietic progenitor cells (CD34+) and their derivatives, and on gene delivery to the liver. In both settings, in vitro and in vivo, SV40 has been very effective. It is therefore a highly promising gene delivery vehicle that may complement other vectors that are currently in use or that are being developed.

Infection with vaccinia virus alters regulation of cell cycle progression.

The effect of vaccinia virus (VV) on cell cycle progression and its regulators was studied. Infected cultures showed significantly increased transit through G1, decreasing the percentage of cells in G1 and increasing the percentage in S phase. The numbers of cells in G2/M were not affected. Because of the increased S-phase fraction at the expense of G1, expression of cyclins and cyclin-dependent kinases (Cdks) that regulate cell cycle checkpoints was examined. Transcripts for cyclins A and B, Cdk2, and Cdc2 were decreased in VV-infected cells as infection progressed. The amounts of p53 and p27 proteins decreased after 12 and 24 h of infection, respectively. The Cdc2 and Cdk2 protein levels were decreased with increasing time after infection. Taken together, these findings would be expected to lead to more cells in S phase and G2/M, as was observed. Therefore, VV actively modulates expression of cellular regulators of the cell cycle and alters cell cycle progression.

Viral vectors for gene therapy: past, present and future.

Gene delivery has been attempted in both experimental and clinical settings. These studies have shown that therapeutic gene transfer is possible, but it has not yet arrived as a practicable therapeutic intervention. This is due in large part to the inability of the vectors used to convey genetic material to a desired location in sufficient quantity and for long enough time to be effective. Current research on viral vectors for gene therapy has focused on reengineering viruses currently being tested as delivery agents, modifying the host to facilitate viral gene transfer and developing new viruses for use in gene transfer. It is too early to know which of these approaches will be effective; however, these ongoing studies are likely to make available in the future an array of gene delivery vehicles with different strengths and weaknesses. It is reasonable to expect that several of the vectors now being studied will prove useful for some therapeutic applications.

Cutaneous Hodgkin's disease.

Involvement of the skin in advanced Hodgkin's disease is well known. However, Hodgkin's disease initially seen in the skin is extremely rare. A patient had Hodgkin's disease that was initially seen as a cutaneous lesion on the buttock. Lymphadenopathy developed two months after completion of local radiotherapy to the cutaneous lesion and seven months after initial diagnosis. The histologic changes in both instances were those of Hodgkin's disease. We discuss problems in the diagnosis and treatment of skin involvement with Hodgkin's disease.

Immunologic consequences of exogenous surfactant administration.

In conclusion, we have shown that human surfactant is immunogenic and that circulating surfactant-antisurfactant immune complexes are detectable in the plasma from infants and in adults with RDS. We found these immune complexes regardless of whether exogenous surfactant was used in the individual treatment regimen. These immune complexes do not yet seem to cause disease in the short term. Long-term effects, if any, are unknown. Indications for surfactant replacement therapy in neonatal RDS are clear. Trials of exogenous surfactant are just beginning in adult RDS, and potential immunogenicity will be of even greater concern in these patients. In all such situations, potential for side effects must be balanced against therapeutic efficacy and the gravity of the disease. Our data indicate that surfactants, particularly heterologous surfactants, are potent immunogens. One cannot assume that using homologous or heterologous surfactants in patients with RDS will always be immunologically innocuous. Nonetheless, based on present data, moderately long-term follow-up (2 to 4 years), we are encouraged by our observation that no selective adverse effects attributable to human surfactant have been recognized, yet mortality from RDS in infants less than 30 weeks has been nearly cut in half.

Histoplasmosis presenting as the carpal tunnel syndrome.

An otherwise asymptomatic 53 year old woman presented with symptoms typical of median nerve compression in the carpal tunnel. At operation, the tendon sheath was obviously inflamed. The diagnosis of histoplasmosis was made on microscopic examination of the surgical specimen. Cultures of synovial tissue grew H. capsulatum. Review of the literature and of experience at Barnes Hospital indicates that the carpal tunnel syndrome is a very unusual primary manifestation of histoplasmosis. However, as histoplasmosis is a treatable cause of the carpal tunnel syndrome, this diagnosis should be considered in endemic areas.