Prime and boost vaccination against HER2/neu in breast cancer

Breast cancer is the most common cancer among women with approximately 180,000 new cases being diagnosed yearly in the United States (1). HER2/neu gene amplification and subsequent protein overexpression is found in 20–30% of breast cancer patients and can lead to the promotion of various metastasis-related properties (2–4) and/or resistance to cancer therapies such as chemotherapy and radiation (5). ^ The protein product of the HER2/neu gene, p185, is a proven target for immunological therapy. Recently, passive immunotherapy with the monoclonal antibody Trastuzumab® has validated an immunological approach to HER2/neu+ breast cancer. Immunity to HER2/ neu, when found in breast cancer patients, is of low magnitude. Vaccination-induced HER2/neu-specific antibodies and HER2/neu-specific cytotoxic T cells could result in long-lived immunity with therapeutic benefit. Many features of DNA vaccines and attenuated viral vectors may contribute to the efficacy of prime-boost vaccination. In particular, vaccines capable of eliciting strong cell-mediated immunity are thought to hold the greatest promise for control of cancer (6–9). ^ To optimize cellular immunization to HER2/neu in my study, the HER2/neu gene was presented to the immune system using a priming vector followed by a second vector used as the boost. In both animals and humans, priming with DNA and boosting with a poxviruses, vaccinia or canarypox appears to be particularly promising for induction of a broad immune responses (10). ^ I tested three gene vaccines encoding the HER2/neu gene: (1) a plasmid, SINCP, that contains part of the genome of Sindbis virus; (2) Viral Replicon Particles (VRP) of Venezuela Equine Encephalitis virus (VEE) and (3) E1/E2a-deleted human Type 5 Adenovirus. In SINCP and the VRP, the caspid and envelope genes of the virus were deleted and replaced with the gene for HER2/neu. SINCP-neu, VRP- neu and Adeno-neu when used alone were effective vaccines protecting healthy mice from challenge with a breast cancer cell line injected in the mammary fat pad or injected i.v. to induce experimental lung metastasis. However, SINCP-neu, VRP-neu or Adeno-neu when used alone were not able to prolong survival of mice in therapeutic models in which vaccination occurred after injection of a breast cancer cell line. ^ When the vaccines were combined in a mixed regimen of a SINCP- neu prime VRP-neu or Adeno-neu boost, there was a significant difference in tumor growth and survival in the therapeutic vaccine models. In vitro assays demonstrated that vaccination with each of the three vaccines induced IgG specific for p185, the gene product of HER2/neu, induced p185-specific T lymphocytes, as measured by tetramer analysis. Vaccination also induced intracellular INF-γ and a positive ELISPOT assay. These findings indicate that SINCP-neu, VRP-neu and Adeno-neu, used alone or in combination, may have clinical potential as adjuvant immunotherapy for the treatment of HER2/neu-expressing tumors. ^

Susceptibility to the biological effects of polyaromatic hydrocarbons is influenced by genes of the major histocompatibility complex

Polyaromatic hydrocarbons are ubiquitous environmental chemicals that are important mutagens and carcinogens. The purpose of this study was to determine whether genes within the major histocompatibility complex (MHC) influence their biological activities. Cell-mediated immunity to dimethylbenz(a)anthracene (DMBA) was investigated in congenic strains of mice. On three different backgrounds, H-2k and H-2a haplotype mice developed significantly greater contact-hypersensitivity responses to DMBA than H-2b, H-2d, and H-2s mice. In B10.A(R1) mice, which are Kk and Id, a vigorous contact-hypersensitivity response was present, indicating that the response was governed by class I, rather than class II, MHC genes. C3H/HeN (H-2k) and C3H.SW (H-2s) strains were also compared for the development of skin tumors and the persistence of DMBA–DNA adducts. When subjected to a DMBA initiation, phorbol 12-tetradecanoate 13-acetate (TPA)-promotion skin-tumorigenesis protocol, C3H/HeN mice, (which develop cell-mediated immunity to DMBA) were found to have significantly fewer tumors than C3H.SW mice (a strain that failed to develop a cell-mediated immune response to DMBA). DMBA–DNA adducts were removed more rapidly in C3H/HeN than in C3H.SW mice. The results indicate that genes within the MHC play an important role in several of the biological activities of carcinogenic polyaromatic hydrocarbons. The observations are consistent with the hypothesis that cell-mediated immunity to chemical carcinogens serves to protect individuals by removing mutant cells before they can evolve into clinically apparent neoplasms.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Impacto no sistema imunológico da utilização prolongada de morfina para tratamento da dor

Introdução. Apesar das evidências dos efeitos imunomodulatórios da morfina, não há na literatura estudos que tenham comparado a interação entre citocinas, imunidade celular (linfócitos T, B e NK) e a administração prolongada de morfina administrada pelas vias oral ou intratecal em doentes com dor crônica neuropática não relacionada ao câncer. Foram avaliados de forma transversal e comparativa 50 doentes com diagnóstico de dor lombar crônica e com presença de radiculopatia (dor neuropática) previamente operados para tratar hérnia discal lombar (Síndrome Dolorosa Pós- Laminectomia), sendo 18 doentes tratados prolongadamente com infusão de morfina pela via intratecal com uso de sistema implantável no compartimento subaracnóideo (grupo intratecal); 17 doentes tratados prolongadamente com morfina pela via oral (n=17) e 15 doentes tratados com fármacos mas sem opióides (grupo sem opioide). Foram analisadas as concentração das citocinas IL-2, IL-4, IL-8, TNFalfa, IFNy, IL-5, GM-CSF, IL-6, IL-10 e IL-1beta no plasma e no líquido cefalorraquidiano; imunofenotipagem de linfócitos T, B e células NK e avaliados os Índice de Escalonamento de Opióide (em percentagem de opióide utilizada e em mg), dose cumulativa de morfina (mg), duração do tratamento em meses, dose final de morfina utilizada (em mg), e equivalente de morfina por via oral (em mg). Resultados. Não houve diferença estatisticamente significativa entre o número de linfócitos T, B e NK nos doentes com morfina administrada pelas vias IT, VO e os não usuários de morfina. Houve correlação positiva entre as concentrações de linfócitos T CD4 e o Índice de Escalonamento de Opióide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlação negativa entre as concentrações de células NK (CD56+) e o Índice de Escalonamento de Opióide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlação positiva entre o número de células NK (CD56+) e a dose cumulativa de morfina (em mg) administrada pelas vias intratecal e oral. Houve correlação positiva entre as concentrações de linfócitos T CD8 e a duração do tratamento em meses nos doentes tratados com morfina pela via oral. As concentrações de IL-8 e IL-1beta foram maiores no LCR do que no plasma em todos os doentes da amostra analisada. As concentrações de IFNy no LCR foram maiores nos doentes que utilizavam morfina pela via oral e nos não usuários de morfina do que nos que a utilizavam pela via intratecal. As concentrações de plasmáticas de IL-5 foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos que não a utilizavam. A concentração de IL-5 no LCR correlacionou-se negativamente com a magnitude da dor de acordo com a EVA nos doentes tratados com morfina pelas via oral ou intratecal. Nos doentes tratados com morfina pelas via oral ou intratecal, a concentração de IL-2 no LCR correlacionou-se positivamente com a magnitude da dor de acordo com a EVA e negativamente com o Índice de Escalonamento de Opióide (em % e mg) e a dose cumulativa de morfina (em mg). As concentrações plasmáticas de GMCSF foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos não a utilizavam. A concentração de TNFalfa no LCR nos doentes tratados com morfina pela via intratecal correlacionou-se negativamente com o Índice de Escalonamento de Opióide (em % e mg), a dose cumulativa de morfina (em mg) e dose equivalente por via oral (em mg) de morfina. A concentração plasmática das citocinas IL-6 e IL-10 correlacionou-se negativamente com a duração do tratamento (em meses) nos doentes tratados com morfina administrada pela via oral. O Índice de Escalonamento de Opióide (em mg e %) correlacionou-se negativamente com as concentrações no LCR de IL-2 e TNFalfa nos doentes tratados com morfina administrada pela via intratecal. O Índice de Escalonamento de Opióide (em mg e %) correlacionou-se negativamente com as concentrações no LCR de IL-2 e IL-5 nos doentes tratados com morfina administrada pela via oral. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações de IL-5 e IL-2 no LCR nos doentes tratados com morfina administrada pelas vias oral e intratecal. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações plasmáticas de IL-4 nos doentes tratados com morfina administrada pela via intratecal. Houve correlação negativa entre a intensidade da dor de acordo com a EVA e as concentrações plasmáticas de IL-1beta nos doentes tratados com morfina administrada pela via intratecal. Conclusões: Os resultados sugerem associações entre citocinas e imunidade celular (células T , B e NK) e o tratamento prolongado com morfina administrada pela via oral ou intratecal. Estes resultados podem contribuir para a compreensão da imunomodulação da morfina administrada por diferentes vias em doentes com dor neuropática crônica não oncológica . São necessários mais estudos sobre os efeitos da morfina sobre o sistema imunológico

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

Active and passive immunization against oral Candida albicans infection in a murine model

Background/aims: Clinical and laboratory studies are consistent with a major role for cell-mediated immunity in recovery from oral infection with Candida albicans, but the role of humoral immunity remains controversial. The purpose of this study was to establish the relative contributions of cellular and humoral immunity to protection against oral candidiasis in a murine model, and to determine whether host responses could be enhanced by different immunization strategies. Results: Active oral immunization was protective in BALB/c and CBA/CaH mice, reducing both fungal burden and duration of infection after secondary challenge, whereas systemic immunization failed to protect against subsequent oral challenge. Candida-specific IgM was the predominant antibody detected in serum following both primary and secondary oral challenge; however, Candida-specific salivary IgA was not detectable. Immunization by passive transfer of either lymphocytes or immune serum did not confer any significant protection against oral infection in either susceptible or resistant mouse strain. Conclusion: The data demonstrate a possible role for mucosa-associated immunity following active immunization by the oral route, most likely exerted by local T lymphocytes resident in the oral mucosa, but there was no evidence to support a role for humoral immunity in protection against oral candidiasis.

Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting

Cell-mediated immunity is important for anti-Candida host defence in mucosal tissues. In this study we used cytokine-specific gene knockout mice to investigate the requirement for T helper type 1 (Th1) and Th2 cytokines in recovery from oral candidiasis. Knockout mice used in this study included interleukin-4 (IL-4), IL-10, IL-12p40, interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF). The mice were challenged either orally or systemically with Candida albicans yeasts, and levels of colonization were determined. IL-12p40 knockout mice developed chronic oropharyngeal candidiasis, but were not more susceptible to systemic challenge. On the other hand, TNF knockout mice displayed increased susceptibility to both oral and systemic challenge, but only in the acute stages of infection. TNF apparently has a protective effect in the acute stages of both oral and systemic candidiasis, whereas IL-12p40 is essential for recovery from oral but not systemic candidiasis. The role of IL-12p40, and its relation to T-cell-mediated responses remain to be determined.

The roles of exercise-induced immune system disturbances in the pathology of heat stroke - The dual pathway model of heat stroke

Heat stroke is a life-threatening condition that can be fatal if not appropriately managed. Although heat stroke has been recognised as a medical condition for centuries, a universally accepted definition of heat stroke is lacking and the pathology of heat stroke is not fully understood. Information derived from autopsy reports and the clinical presentation of patients with heat stroke indicates that hyperthermia, septicaemia, central nervous system impairment and cardiovascular failure play important roles in the pathology of heat stroke. The current models of heat stroke advocate that heat stroke is triggered by hyperthermia but is driven by endotoxaemia. Endotoxaemia triggers the systemic inflammatory response, which can lead to systemic coagulation and haemorrhage, necrosis, cell death and multi-organ failure. However, the current heat stroke models cannot fully explain the discrepancies in high core temperature (Tc) as a trigger of heat stroke within and between individuals. Research on the concept of critical Tc: as a limitation to endurance exercise implies that a high Tc may function as a signal to trigger the protective mechanisms against heat stroke. Athletes undergoing a period of intense training are subjected to a variety of immune and gastrointestinal (GI) disturbances. The immune disturbances include the suppression of immune cells and their functions, suppression of cell-mediated immunity, translocation of lipopolysaccharide (LPS), suppression of anti-LPS antibodies, increased macrophage activity due to muscle tissue damage, and increased concentration of circulating inflammatory and pyrogenic cytokines. Common symptoms of exercise-induced GI disturbances include diarrhoea, vomiting, gastrointestinal bleeding, and cramps, which may increase gut-related LPS translocation. This article discusses the current evidence that supports the argument that these exercise-induced immune and GI disturbances may contribute to the development of endotoxaemia and heat stroke. When endotoxaemia can be tolerated or prevented, continuing exercise and heat exposure will elevate Tc to a higher level (> 42 degrees C), where heat stroke may occur through the direct thermal effects of heat on organ tissues and cells. We also discuss the evidence suggesting that heat stroke may occur through endotoxaemia (heat sepsis), the primary pathway of heat stroke, or hyperthermia, the secondary pathway of heat stroke. The existence of these two pathways of heat stroke and the contribution of exercise-induced immune and GI disturbances in the primary pathway of heat stroke are illustrated in the dual pathway model of heat stroke. This model of heat stroke suggests that prolonged intense exercise suppresses anti-LPS mechanisms, and promotes inflammatory and pyrogenic activities in the pathway of heat stroke.

Cytokine-specific Gene-knockout Studies in Oropharyngeal Candidiasis

Oropharyngeal candidiasis is a common clinical problem encountered in patients with defects in innate or cell-mediated immunity. We have previously shown that recovery from chronic oropharyngeal candidiasis is dependent on CD4+ T-cell augmentation of neutrophil and macrophage candidacidal activity, and that the immune response is characterised by the production of cytokines such as IL-12 and IFN-gamma by cells in the local draining lymph nodes, and by the expression of TNF-alpha in the oral tissues. Objective: The purpose of this study was to elaborate on the role of these cytokines in recovery from oropharyngeal candidiasis, by using cytokine-specific gene-knockout mice. Methods: These mice are created by targeted gene mutation (tm1) of embryonic stem (ES) cells microinjected into host embryos. IL-4, IL-10, IL-12, IFN-gamma and TNF-alpha knockout mice, and appropriate controls, were infected orally with 108 viable C. albicans yeasts. The infection was quantified by swabbing the oral cavity and plating on Sabouraud's agar. Results: Tnftm1mice developed an acute severe infection characterized by an increased fungal load in the early stages of infection, but cleared the yeast within the same time frame as control mice (21 days). On the other hand, Il12btm1 mice developed a chronic oropharyngeal infection (120 days) similar to that seen in T-cell deficient (Foxn1nu/Foxn1nu) mutant mice. There was no significant difference between Il4tm1, Il10tm1, and Ifngtm1 mice and their respective controls. Conclusions: Tnftm1 mice may be rendered more susceptible through impaired recruitment of phagocytic cells, and/or impaired killing of C. albicans, whereas Il12btm1 mice may not be capable of activating naïve T-cells or inducing an appropriate cellular immune response. Supported by NHMRC and ADRF.

Immunological studies in multiple low dose streptozotocin induced diabetes in mice

1. Multiple low doses of streptozotocin (MSZ) treatment successfully induced diabetes in male TO, MFI and HO lean mice. In contrast however, BALB/c mice failed to develop persistent hyperglycaemia. Single streptozotocin (SSZ) treatment also produced diabetes in TO mice. SSZ treatment however, produced severe weight loss and atrophy of the lymphoid organs. MSZ treatment on the other hand, was not cytotoxic towards lymphoid organs and, whilst there was no loss of body weight, growth rates were reduced in MSZ treated mice. 2. Following sheep red blood cell (SRBC) immunisation of MSZ-treated mice, haemagglutination titres, and numbers of antigen reactive cells and plaque forming cells were all significantly lower than control values. 3. In vitro proliferation of spleen cells in response to phytohaemagglutinin (PHA) and conconavalin A (ConA) was found to be significantly depressed in MSZ treated mice. However, T-lymphocyte responses were intact when the mice were not overtly hyperglycaemic. In contrast, however, T cell independent responses to lipopolysaccharide (LPS) were generally intact throughout the study period. 4. Cell mediated immunity, as assessed by measurements of delayed (Type IV) hypersensitivity, was also depressed in MSZ treated mice. This suppression could be reversed by insulin therapy. 5. Both natural killer cell activity and antibody dependent cell mediated cytotoxicity were found to be significantly increased in MSZ treated mice. 6. Histological examination of the pancreas showed the presence of insulitis, in MSZ treated mice, and cytotoxic effector cells against obese mice islet cells (as assessed by 51Cr release) and HIT-T15 cells (as assessed by insulin secretion) were found to be significantly increased. Furthermore, these effector cells were also found to show increased proliferation in the presence of homogenates prepared from HIT-T15 cells. Examination of the Sera from MSZ treated mice showed that islet cell surface antibodies were present.

Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants

The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.

Nanotechnology for the delivery of vaccines

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris): Morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n = 190) and manatees (n = 56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immunocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included T$\rm\sb{h}$, T$\rm\sb{c/s}$, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating T$\rm\sb{h}$ cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris) : morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n=180) and manatees (n=56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immuocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included Th, Tes, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating Th cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.