Aerosol delivery of synthetic DNA containing CpG motifs in broiler chicks at hatch under field conditions using a commercial-scale prototype nebulizer provided protection against lethal Escherichia coli septicemia.

Synthetic DNA containing CpG motifs (CpG-ODN) are potent innate immune stimulators in neonatal and adult broiler chickens against bacterial septicemia. We have recently demonstrated that intrapulmonary (IPL) delivery of CpG-ODN as microdroplets under laboratory conditions can protect neonatal chickens against lethal Escherichia coli septicemia. The objectives of this study were to develop a commercial-scale poultry nebulizer (CSPN) that can deliver CpG-ODN as microdroplets in neonatal broiler chicks in the hatcheries and study the efficacy of CSPN in inducing immune-protective effects under different environmental conditions in 2 geographical locations in Canada. Three field experiments were conducted in commercial poultry hatcheries during different seasons of the year in Saskatchewan and British Columbia, Canada. Neonatal broiler chicks (n = 8,000/experiment) received CpG-ODN by the IPL route in the CSPN chamber for 30 min, and control chicks received distilled water (DW) for 30 min. Broiler chicks (CpG-ODN-240 chicks/experiment and DW-40 chicks/experiment) were randomly sampled from all locations of the CSPN after nebulization and challenged with a lethal dose of E. coli to examine the CpG-ODN nebulization induced protection. We found a significant level (P < 0.05) of protection in broiler chicks against E. coli challenge, suggesting that the newly built CSPN successfully delivered CpG-ODN via the IPL route. We found that when the CSPN was maintained at humidex 28°C or below and relative humidity (RH) between 40 and 60%, neonatal birds were significantly (P < 0.05) protected against E. coli septicemia after IPL delivery of CpG-ODN. By contrast, protection in chicks was adversely affected when the CSPN was maintained at the humidex of 29°C or higher and RH of 70%. Overall, the present study successfully built a CSPN for CpG-ODN delivery in chicks at the hatchery and revealed that the temperature, humidity, and humidex were critical parameters in CSPN for efficient delivery of CpG-ODN.

Male eyespan size is associated with meiotic drive in wild stalk-eyed flies (Teleopsis dalmanni).

This study provides the first direct evidence from wild populations of stalk-eyed flies to support the hypothesis that male eyespan is a signal of meiotic drive. Several stalk-eyed fly species are known to exhibit X-linked meiotic drive. A recent quantitative trait locus analysis in Teleopsis dalmanni found a potential link between variation in male eyespan, a sexually selected ornamental trait, and the presence of meiotic drive. This was based on laboratory populations subject to artificial selection for male eyespan. In this study, we examined the association between microsatellite markers and levels of sex ratio bias (meiotic drive) in 12 wild T. dalmanni populations. We collected two data sets: (a) brood sex ratios of wild-caught males mated to standard laboratory females and (b) variation in a range of phenotypic traits associated with reproductive success of wild-caught males and females. In each case, we typed individuals for eight X-linked microsatellite markers, including several that previously were shown to be associated with male eyespan and meiotic drive. We found that one microsatellite marker was very strongly associated with meiotic drive, whereas a second showed a weaker association. We also found that, using both independent data sets, meiotic drive was strongly associated with male eyespan, with smaller eyespan males being associated with more female-biased broods. These results suggest that mate preference for exaggerated male eyespan allows females to avoid mating with males carrying the meiotic drive gene and is thus a potential mechanism for the maintenance and evolution of female mate preference.

Effect of topical interferon-γ gene therapy using gemini nanoparticles on pathophysiological markers of cutaneous scleroderma in Tsk/+ mice.

Scleroderma is a chronic disorder manifested by excessive synthesis and deposition of collagen in skin and connective tissue, vascular abnormalities, and autoimmunity. Using microarray and real-time PCR data, we show that intradermally expressed interferon γ (IFN-γ), generated after intradermal injection of IFN-γ-coding plasmid, and non-invasive topical nanoparticle (TNP) treatment with IFN-γ-coding plasmid, decreased collagen synthesis (via the Jak/Stat 1 pathway), upregulated Th1 cytokine levels, and downregulated the profibrotic cytokine Transforming growth factor ß and the Smad pathways in the Tsk/+ (tight-skin scleroderma) mouse model. The TNP gene delivery system was constructed from gemini surfactant 16-3-16 and IFN-γ-coding plasmid. Topical administration of IFN-γ-coding plasmid in TNPs was effective in expressing IFN-γ levels after a 20-day treatment regimen without increased TLR4, CCL2, CCL11 and CCR2 mRNA levels that were observed in injected animals, signs considered to be innate responses to injury. The more uniform transgene IFN-γ expression caused significant (70-72%) collagen reduction, as assessed by reverse transcription real-time PCR. These results demonstrate efficient in vivo transfection using a gemini surfactant-based TNP delivery system able to modulate excessive collagen synthesis in scleroderma-affected skin.

Pathogenesis and therapeutic approaches for improved topical treatment in localized scleroderma and systemic sclerosis.

SSc is a chronic progressive disorder of unknown aetiology characterized by excess synthesis and deposition of collagen and other extracellular matrix components in a variety of tissues and organs. Localized scleroderma (LS) differs from SSc in that with LS only skin and occasionally subcutaneous tissues are involved. Although rarely life threatening, LS can be disfiguring and disabling and, consequently, can adversely affect quality of life. There is no known effective treatment for LS, and various options, including, as examples, corticosteroids and other immunomodulatory agents, ultraviolet radiation and vitamin D analogues, are of unproven efficacy. Clinical trials evaluating combination therapy such as corticosteroids with MTX or UVA1 exposure with psoralens have not been established as consistently effective. New immunomodulators such as tacrolimus and thalidomide are also being evaluated. A better understanding of the molecular and cellular mechanisms of LS has led to evaluation of new treatments that modulate profibrotic cytokines such as TGF-beta and IL-4, regulate assembly and deposition of extracellular matrix components, and restore Th1/Th2 immune balance by administering IL-12 or IFN-gamma. IFN-gamma acts by directly inhibiting collagen synthesis and by restoring immune balance. In this review, we evaluate current and future treatment options for LS and cutaneous involvement in SSc. Recent advances in therapy focus mainly on anti-fibrotic agents. Delivery of these drugs into the skin as the target tissue might be a key factor in developing more effective and safer therapy.

Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse model.

The nasal mucosa is an important arm of the mucosal system since it is often the first point of contact for inhaled antigens. The ineffectiveness of the simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies in appropriate delivery systems and adjuvants. We have evaluated biphasic lipid vesicles as a novel intranasal (i.n.) delivery system (designated as vaccine targeting adjuvant, VTA) containing bacterial antigens and CpG oligodeoxynucleotides (ODNs). Results show that administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in greater induction of IgA levels in serum (P< 0.05) and mucosal antibody responses such as IgA in nasal secretions and lung (P< 0.01) after immunization with a combined subcutaneous (s.c.)/i.n. as compared to s.c./s.c. approach. Based on antibody responses, VTA formulations were found to be suitable as delivery systems for antigens and CpG ODNs by the intranasal route, resulting in a Th2-type of immune response, characterized by IgG1 and IL-4 production at the systemic level.

Efficacy of the antiadhesin octyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-4)-2-O-propyl-beta-D-galactopyranoside (Fimbrigal-P) in a rat oral candidiasis model.

Adherence of Candida albicans to buccal epithelial cells via its fimbrial subunit requires the minimal disaccharide sequence beta-GalNAc(1-4)-beta-galactosidase in host cell receptors asialo-GM1 or asialo-GM2. This and other disaccharides and some of its synthetic derivatives have been shown to inhibit purified fimbrial or pathogen binding in vitro. This study evaluates the in vivo efficacy of the propyl derivative of this disaccharide, octyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-4)-2-O-propyl-beta-D-galactopyranoside, or Fimbrigal-P, incorporated into a mucoadhesive polymer formulation in a rat oral candidiasis model. Colony counts of microcurette samples from the oral cavity and tongue homogenates were used to estimate the effectiveness of four treatment modalities to reduce oral fungal burden. All treatment modalities (preventative, premixing with the Candida inoculant, drinking water, and treatment) significantly reduced fungal burden compared to untreated control animals by day 9; however, the preventative and pre-mixing approaches provided a faster rate of fungal clearance. The low toxicity and immunogenicity of this synthetic carbohydrate and its stability in saliva, as demonstrated by high-performance liquid chromatography, make it a promising candidate for the prevention and treatment of microbial infections in which the pathogen relies on the beta-GalNAc(1-4)-beta-galactosidase disaccharide to establish adherence.

Induction of protective immunity in pigs after immunisation with CpG oligodeoxynucleotides formulated in a lipid-based delivery system (Biphasix).

A large number of studies demonstrated the immunostimulatory effects of CpG oligonucleotides (ODN), particularly in mice. In the present study, we evaluated the ability of lipid-based delivery systems to enhance the adjuvant effect of CpG-ODN and protect against infection in a porcine pleuropneumonia model. Increased levels of OmlA-specific antibody were detected in animals immunised with OmlA and CpG-ODN formulated in the delivery system Biphasix-vaccine targeting adjuvant (VTA), compared to pigs immunised with VTA without CpG-ODN or CpG-ODN alone. In addition, the responses induced by VTA/CpG formulation were similar to those induced by the commercial adjuvant VSA; however, VTA formulations caused significantly less tissue damage than VSA.

Targeting of liposomes to human keratinocytes through adhesive peptides from immunoglobulin domains in the presence of IFN-gamma.

T-cell adhesion is often dictated by the presence of intercellular adhesion molecule-1 (ICAM-1) on the target cell surface. Reconstitution of P(0) protein into liposomes increases adhesion to melanoma cells expressing ICAM-1. In our study, the effect of peptides derived from P(0) protein and leukocyte function associated-antigen 1 (LFA-1) on IFN-gamma-stimulated human keratinocytes was investigated. Covalently linked P(0)-peptide-1, from the Ig-like domain, increased specific liposome binding to IFN-gamma-stimulated keratinocytes in a dose-dependent manner. C-terminal-derived P(0)-peptide-3 increased liposome binding nonspecifically. LFA-1 and RGD peptides had no apparent effect. P(0)-peptide-1 is thus a potential targeting ligand for liposomal drug delivery to ICAM-1 expressing keratinocytes in inflammatory dermatoses.

Targeting of liposomes to melanoma cells with high levels of ICAM-1 expression through adhesive peptides from immunoglobulin domains.

The P(0) protein is an immunoglobulin [Ig] superfamily cell adhesion molecule from peripheral nerve myelin. Synthetic peptides derived from the P(0) protein and leukocyte function-associated antigen-1 (LFA-1) were investigated as potential ligands for targeting liposomes to intercellular adhesion molecule-1 (ICAM-1) expressing melanoma cells. Three synthetic P(0) peptides and one LFA-1 peptide were selected for linkage to liposome surfaces. P(0)-peptide-1, from the extracellular Ig-like domain, increased liposome binding to M21 (6.36-fold) and A-375 (1.85-fold) cells compared to control blank liposomes, but did not increase liposome binding to MeM 50-10 cells. P(0)-peptide-3, from the basic intracellular domain, increased binding of liposomes to all three melanoma cell lines nonspecifically due to its high content of positively charged amino acids. LFA-1- and negative control arg-gly-asp (RGD)-peptides did not affect liposome binding to M21 cells. The extent of P(0)-peptide-1-liposome binding to human melanoma cell lines correlated with the level of cellular ICAM-1 expression (r(2) = 0.868). P(0)-peptide-1-mediated targeting of liposomes might, therefore, prove useful in the development of drug delivery systems for treatment of ICAM-1 expressing malignant melanomas.

Randomized trial of progressive resistance training to counteract the myopathy of chronic heart failure.

Chronic heart failure (CHF) is characterized by a skeletal muscle myopathy not optimally addressed by current treatment paradigms or aerobic exercise. Sixteen older women with CHF were compared with 80 age-matched peers without CHF and randomized to progressive resistance training or control stretching exercises for 10 wk. Women with CHF had significantly lower muscle strength (P < 0.0001) but comparable aerobic capacity to women without CHF. Exercise training was well tolerated and resulted in no changes in resting cardiac indexes in CHF patients. Strength improved by an average of 43.4 +/- 8.8% in resistance trainers vs. -1.7 +/- 2.8% in controls (P = 0.001), muscle endurance by 299 +/- 66% vs. 1 +/- 3% (P = 0.001), and 6-min walk distance by 49 +/- 14 m (13%) vs. -3 +/- 19 m (-3%) (P = 0.03). Increases in type I fiber area (9.5 +/- 16%) and citrate synthase activity (35 +/- 21%) in skeletal muscle were independently predictive of improved 6-min walk distance (r2 = 0.78; P = 0.0024). High-intensity progressive resistance training improves impaired skeletal muscle characteristics and overall exercise performance in older women with CHF. These gains are largely explained by skeletal muscle and not resting cardiac adaptations.

Non-invasive administration of drugs through the skin: challenges in delivery system design.

Vehicles designed to enhance drug delivery through the skin must incorporate specific elements that improve the ability of the delivery system to overcome the barrier posed by the stratum corneum. This review discusses several chemical penetration enhancers that have been investigated as potential tools to increase drug flux. In addition, lipid-based delivery systems offer an attractive alternative to traditional drug vehicles. The relationship between liposome composition and drug permeation is discussed, in addition to the possible mechanism of action of lipid vesicle-mediated drug delivery.

Vaccine delivery: lipid-based delivery systems.

Needle-free delivery of vaccines should not only increase compliance, but should also prove to be a safer and less traumatic method of vaccine delivery. One of the potential ways to achieve needle-free delivery is with the use of lipid-based delivery systems. To demonstrate the utility of these systems, we have shown them to be effective with proteins produced by recombinant DNA technology, plasmid-based vaccines, as well as conventional vaccines. Furthermore, these lipid-based delivery systems were shown to be effective in inducing mucosal immunity if delivered to mucosal surfaces or systemic immunity if different transdermally. These approaches have the potential to revolutionize vaccine delivery in humans and animals.

Association of muscle power with functional status in community-dwelling elderly women.

BACKGROUND: Identification of the physiologic factors most relevant to functional independence in the elderly population is critical for the design of effective interventions. It has been suggested that muscle power may be more directly related to impaired physical performance than muscle strength in elderly persons. We tested the hypothesis that peak muscle power is closely associated with self-reported functional status in sedentary elderly community-dwelling women. METHODS: We used baseline data that were collected as part of a 1-year randomized controlled clinical trial of a combined program of strength, power, and endurance training in 80 elderly women (mean age 74.8 +/- 5.0 years) with 3.2 +/- 1.9 chronic diseases, selected for baseline functional impairment and/or falls. RESULTS: Functional status at baseline was related in univariate analyses to physiologic capacity, habitual physical activity level, neuropsychological status, and medical diagnoses. Leg power had the strongest univariate correlation to self-reported functional status (r = -.47, p < .0001) of any of the physiologic factors we tested. In a forward stepwise regression model, leg press power and habitual physical activity level were the only two factors that contributed independently to functional status (r = .64, p < .0001), accounting for 40% of the variance in functional status. CONCLUSIONS: Leg power is a strong predictor of self-reported functional status in elderly women.

Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery.

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eradicated. However, despite these successes there are still many microbial diseases that cause tremendous suffering because there is no vaccine or the vaccines available are inadequate. In addition, even if vaccines were available for all infectious diseases there is no guarantee that people would use them routinely. One of the major impediments to ensuring vaccine efficacy and compliance is that of delivery. Presently most vaccines are given by intramuscular administration. Unfortunately this is often traumatic, especially in infants. Thus, if it was possible to replace intramuscular immunization by mucosal (oral/intranasal) or transdermal delivery it may be possible to both enhance mucosal immunity as well as improve overall compliance rates. The transdermal route has been used by the pharmaceutical industry for the delivery of various low molecular weight drugs. Some of the approaches used for smaller compounds may also have potential for delivery of either protein or polynucleotide vaccines. However, there is a greater challenge to delivering large molecular weight molecules through the skin due to size, charge and other physicochemical properties. This review will describe the recent advances that have been made in dermal and topical delivery as related to vaccines.

Effects of IL-12 on immune responses induced by transcutaneous immunization with antigens formulated in a novel lipid-based biphasic delivery system.

The development of non-invasive methods for the delivery of proteins through the permeability barriers, such as the intact skin, will greatly facilitate the administration of human and veterinary vaccines. In the present study we used recombinant Pasteurella haemolytica leukotoxin (Lkt) and hen egg lysozyme (HEL) as model antigens to investigate the ability of transdermal administration of vaccine antigens to induce humoral and cellular responses in mice and to assess the immunomodulatory effects of IL-12 on these antigen-specific immune responses. Mice were immunized by the transdermal route with Lkt or HEL formulated in a novel lipid-based biphasic delivery system (BPDS). Transdermal delivery of Lkt or HEL induced strong polarized Th2 responses characterized by enhancement of antigen-specific IgG1 antibody subclass and predominant induction of antigen specific IL-4 over IFN-gamma in spleen and draining lymph nodes cells. Animals immunized by topical application of formulations containing antigen and IL-12 developed significantly lower antibody titres without significant changes in IL-4 or IFN-gamma secreting cells (SC) in the draining lymph nodes or spleen cells. Our results indicated that application of antigens formulated in BPDS induced antigen-specific immune responses. Furthermore, incorporation of IL-12 to the vaccine formulation influences the induction of antibody responses induced by transdermal immunization. We demonstrated the feasibility of using this technology for the development of non-invasive methods of vaccine administration.

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses.

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.

Transtracheal administration of interleukin-12 induces neutrophil responses in the murine lung.

Although the roles of interleukin-12 (IL-12) in the immunomodulation of antigen-specific responses are well characterized, the effects of IL-12 on the respiratory tract following mucosal administration are not well defined. Therefore, we investigated changes in the murine lung shortly after intranasal (i.n.) administration of murine IL-12. We showed that IL-12 induced neutrophil influx to the murine lung in both C57BL/6 and BALB/c mice. Histologic examination revealed that intranasal administration of IL-12 with liposomes induced focal neutrophil infiltration into the alveoli and a significant increase in neutrophils in bronchoalveolar lavage fluids when compared with administration of liposomes alone. In vitro chemotaxis assays indicated that the observed pulmonary neutrophil response induced by IL-12 could have been due in part to the direct chemotactic activity of IL-12 for murine neutrophils.

Acute vaginal candidosis model in the immunocompromized rat to evaluate delivery systems for antimycotics.

A rat model of vaginal candidosis, suitable for screening new antifungal compounds or novel formulations was developed and validated. Two strains of Candida albicans, different inoculation schedules, quantitative assays for fungal burden and the necessity for immunosuppression were tested. The infection was evaluated clinically and histologically by fungal counts from the vaginal discharge. The animals without immunosuppression did not sustain the infection beyond 2-3 days, however, after immunosuppression with cyclophosphamide, the infection can be maintained for about 2 weeks and is suitable for the testing of topical antifungal therapies.

Dermal and transdermal delivery of protein pharmaceuticals: lipid-based delivery systems for interferon alpha.

The dermal and transdermal delivery of protein pharmaceuticals faces enormous challenges, and at the same time has very significant potential for the non-invasive treatment of both localized and systemic diseases. In this article we review the various approaches used to enhance and control the delivery of protein therapeutic agents through the dermal barrier. We show results of the delivery of interferon (IFN) alpha, an antiviral agent used in the treatment of condylomata acuminata (genital warts), using lipid-based delivery systems (LBDS). In the general category of LBDS, we investigated the use of liposomes and fatty acylation as ways to increase IFNalpha delivery into human skin.

Polynucleotide vaccines: potential for inducing immunity in animals.

Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.