An antisense oligonucleotide targeting the growth hormone receptor inhibits neovascularization in a mouse model of retinopathy.

PURPOSE: We have demonstrated that a 2'-O-methoxyethyl modified antisense oligonucleotide against the mouse growth hormone (GH) receptor (GHr) reduces GH binding and serum insulin-like growth factor-1 in normal mice. We tested whether this systemically delivered antisense oligonucleotide could inhibit neovascularization in mice with oxygen induced retinopathy (OIR). METHODS: OIR was induced in C57BL/6 mice by housing them in 75% oxygen across postnatal days (P)7 to 12 followed by five days in room air. Shams were in room air from P0-17. GHr antisense oligonucleotide, ATL 227446, was administered by early (P7, 8, 9, 11, 13, 15, and 17) or late (P12-16) intervention at doses of 5, 10, 20, and 30 mg/kg. Other mice were treated with either vehicle (saline), the somatostatin analog octreotide (20 mg/kg/bi-daily), or control oligonucleotides ATL 261303 (at 20 mg/kg by late and early intervention) or ATL 260120 (at 20 and 30 mg/kg by early intervention only). Blood vessel profiles were counted in 3 mm paraffin sections of inner retina. RESULTS: OIR increased blood vessel profiles by 2.5 fold compared to shams. In OIR, early intervention GHr antisense oligonucleotide ATL 227446 reduced blood vessel profiles at higher doses including 10 mg/kg, and 30 mg/kg resulted in the greatest reduction (38%). In OIR, late intervention with all doses of GHr antisense oligonucleotide ATL 227446 reduced blood vessel profiles to a similar extent, and the highest dose resulted in a 26% reduction compared to OIR. Octreotide reduced blood vessel profiles in OIR mice by 26%. In OIR, ATL 261303 had no effect on blood vessel profiles, while 30 mg/kg ATL 260120 reduced blood vessel profiles by 18%. CONCLUSIONS: Systemically delivered antisense oligonucleotides directed against the GHr are a potential novel treatment for ocular neovascularization related disorders.

Claims of current inhaled corticosteroid patents for treating asthma.

The advent and evolution of corticosteroid treatment strategies over the preceding decades means that asthma is now at least controllable for the majority of asthmatics. The main mode of action for corticosteroids is the inhibition of the nuclear factor-kappaB (NF-kappaB) pathway which dampens the pulmonary inflammatory response associated with asthma pathology. The effectiveness of these drugs and the growing market means that there is strong competitive pressure for pharmaceutical companies to improve, or at the very least maintain, their intellectual property position in corticosteroid treatments for asthma. The most notable feature of the intellectual property situation for inhaled corticosteroids is the impending expiry of a large raft of patents associated with many of the market leading drugs. As efficacy of inhaled drugs is intimately related to how effectively the drug is delivered to the lung, there are a variety of options available for patent protection, including protecting the drug formulation itself as well as various components of a compatible delivery device. In the absence of new corticosteroid chemistries, such approaches will provide extended intellectual property protection and assist in the maintenance of market share for many of the leading inhaled corticosteroids.

The ovine nasal mucosa: an alternative tissue site for mucosal immunization.

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.

Antibody isotype profiles in serum and circulating antibody-secreting cells following mucosal and peripheral immunisations of sheep.

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.

Alternative routes of mucosal immunization in large animals.

Mucosal immunization regimes that employ the oral route of delivery are often compromised by antigen degradation in the stomach. Moreover, tolerance or immunological unresponsiveness to orally delivered vaccine antigens is also a major problem associated with this route of immunization. Immunization by alternative routes including intrarectal (i.r.) and intranasal (i.n.) is becoming increasingly recognized in large animals for generating protective antibody responses at mucosal surfaces. These approaches are particularly useful in ruminant species which have four stomachs that can potentially interfere with antigen presentation to mucosal inductive sites of the gut. Modifications to enhance existing mucosal immunization regimes have also been explored through the use of alternative antigen delivery systems and mucosal adjuvants. The combination of alternative immunization routes and the use of appropriate antigen delivery systems appear to be a rational approach for providing protective immunity at mucosal surfaces. There has been a considerable amount of research conducted on evaluating the efficacy of emerging antigen delivery systems and novel adjuvants for improved immunity to mucosal immunization but very little of this work has been specific to the mucosal compartment of large animals. The aim of this review is therefore to assess the feasibility and practicality of using large animals (particularly sheep, cattle and pigs) for inducing and detecting specific immune responses to alternative mucosal routes of immunization.

Optimization of an ovine antibody-secreting cell assay for detection of antigen-specific immunoglobulin production in peripheral blood leukocytes.

Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.

Immunological aspects of controlled antigen delivery.

Recent advances in controlled delivery systems for protein pharmaceuticals such as microspheres, liposomes, pumps and implants, have provided a new avenue for delivery of vaccine antigens. There has, however, been considerable confusion over the way in which continuous antigen delivery affects the outcome of an immune response. To date, there has been little systematic study of the influence of varying antigen exposure times and release profiles on the phenotype of the immune response, or indeed the balance between immunity and tolerance as most studies have concentrated on optimising responses to a particular antigen of interest in a single model system. As these delivery systems would find particular advantages in management of livestock species, where the use of a single administration vaccine would significantly enhance management practices, it is important to understand the relationship between controlled antigen delivery and immunity. This paper describes how existing controlled antigen delivery studies have contributed to our understanding of the development of the immune response and demonstrates how continuous antigen delivery is useful, and possibly advantageous in the generation of immunity, the maturation of the immune response and the extension of the effector response.

Manipulating the immune response; applications in livestock breeding.

There are many opportunities for the use of immune modulation techniques in livestock that offer the potential to reduce the requirements for chemical usage and surgical intervention in standard management practices. While vaccination has been used for many years for disease control, there are areas in which vaccination has not been very successful, including the induction of mucosal responses, the induction of cellular responses, and the ability to induce extended duration of protection after a single administration of antigen. In addition, new areas of immunological intervention such as immunisation against reproductive hormones offer new opportunities to modify not only reproductive performance, but also growth, metabolism, carcass quality and behaviour in livestock. These new techniques bring increased need for enhanced efficacy and duration of response. While extensive studies in vaccination have shown that many of the desired immunological responses can be induced in experimental conditions, effective application in the field is dependent upon the development of vaccine delivery methods that are practical within the confines of an effective livestock management system. This paper outlines restrictions that may be imposed on vaccine delivery to livestock and introduces controlled antigen delivery as a potential method for single dose vaccination.

Cellular and molecular characterisation of the ovine rectal mucosal environment.

Immunohistological characterisation of ovine rectal tissue has revealed the presence of lymphoid follicles, predominantly in the submucosa, that closely resemble those found in intestinal Peyer's patches (PPs). Distinct T (CD4+, CD8+, gammadelta-TCR+) and B (CD21+, CD45R+) lymphocyte staining patterns were observed within and around follicles of the rectal mucosa. In addition, IgA+ and IgE+ cells were also found at this tissue site, with both phenotypes commonly residing in the lamina propria. RT-PCR examination of the cytokines expressed in the rectal mucosal tissue revealed consistently high levels of TGFbeta and IL-8 mRNA, low levels of IL-2 mRNA and no detectable IL-4 mRNA. The presence of lymphoid follicles, IgA+ plasma cells and IgA-inducing cytokines in rectal tissue of sheep indicate that this may be a suitable route for delivering mucosal vaccines.