Die Synthese von Mucin-Glycopeptid-Vakzinkonjugaten und Adjuvanzien für eine potentielle Krebsimmuntherapie

Die vorliegende Arbeit beschäftigt sich mit der Synthese von unterschiedlichen tumorassoziierten Antigenen, die in Form von glycosylierten Aminosäurebausteinen durch Festphasensynthese in den tandem repeat des epithelialen Mucin MUC1 eingebaut wurden. Zur Erzeugung von selektiven Immunantworten wurden synthetische MUC1-Glycopeptide über einen nicht immunogenen Spacer mit einer Reihe von Immunstimulanzien umgesetzt. Es wurden hierbei zwei Ziele verfolgt. Zum einen sollten mit Hilfe von synthetischen MUC1-Glycopeptiden und Immunstimulanzien cytotoxische T-Zellen ausgebildet werden, die Tumorzellen mit überexprimiertem MUC1 am Wachstum hindern und sie in den programmierten Zelltod treiben. Zum anderen sollte mit Hilfe von TH2-Epitopen eine adaptive humorale B-Zell-vermittelte Immunantwort in Form von Antikörpern ausgelöst werden. Zur Ausbildung von cytotoxischen T-Zellen kamen Vakzinkonjugate zum Einsatz, die aus dem IL-1β(163 – 169), dem TLR2-Agonisten (Pam3Cys-Ser-Lys4) oder dem TLR4-Agonisten (spacergebundenes MPL A) bestehen und jeweils an MUC1-Glycopeptide konjugiert wurden. Darüber hinaus wurde eine neue moderne Syntheseroute für das MPL-A entwickelt, welches als Adjuvans in den verschiedenen Vakzinen, wie Cervarix®, Fendrix® und Stimuvax®, Verwendung findet und die Immunantwort der Vakzinkonstrukte nochmals verstärken sollte. Zur Induktion von Antikörpern kamen die OVA(323 – 339)-, die Tetanus-Toxoid- und die BSA-Konjugate zur Anwendung, an denen MUC1-Glycopeptide angeknüpft wurden.

An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon

A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette–Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette–Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.

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.

Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstrate in vivo protective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response using in vitro assays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this further in vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infected in vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that the in vivo protection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals have in vivo protective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.

Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80).

To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.

Valutazione della risposta immunitaria cellulare e fenotipizzazione linfocitaria nel suino in corso di infezione naturale da virus della PRRS (“Porcine Reproductive and Respiratory Syndrome”) e dopo stimolazione di PBMC (“Peripheral Blood Mononuclear Cells”) in vitro con Peptide Killer (KP)

SUMMARY The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is one of the most spread pathogens in swine herds all over the world and responsible for a reproductive and respiratory syndrome that causes severe heath and economical problems. This virus emerged in late 1980’s but although about 30 years have passed by, the knowledge about some essential facets related to the features of the virus (pathogenesis, immune response, and epidemiology) seems to be still incomplete. Taking into account that the development of modern vaccines is based on how innate and acquire immunity react, a more and more thorough knowledge on the immune system is needed, in terms of molecular modulation/regulation of the inflammatory and immune response upon PRRSV infection. The present doctoral thesis, which is divided into 3 different studies, is aimed to increase the knowledge about the interaction between the immune system and the PRRS virus upon natural infection. The objective of the first study entitled “Coordinated immune response of memory and cytotoxic T cells together with IFN-γ secreting cells after porcine reproductive and respiratory syndrome virus (PRRSV) natural infection in conventional pigs” was to evaluate the activation and modulation of the immune response in pigs naturally infected by PRRSV compared to an uninfected control group. The course of viremia was evaluated by PCR, the antibody titres by ELISA, the number of IFN-γ secreting cells (IFN- SC) by an ELISPOT assay and the immunophenotyping of some lymphocyte subsets (cytotoxic cells, memory T lymphocytes and cytotoxic T lymphocytes) by flow cytometry. The results showed that the activation of the cell-mediated immune response against PRRSV is delayed upon infection and that however the levels of IFN-γ SC and lymphocyte subsets subsequently increase over time. Furthermore, it was observed that the course of the different immune cell subsets is time-associated with the levels of PRRSV-specific IFN-γ SC and this can be interpreted based on the functional role that such lymphocyte subsets could have in the specific production/secretion of the immunostimulatory cytokine IFN-γ. In addition, these data support the hypothesis that the age of the animals upon the onset of infection or the diverse immunobiological features of the field isolate, as typically hypothesized during PRRSV infection, are critical conditions able to influence the qualitative and quantitative course of the cell-mediated immune response during PRRSV natural infection. The second study entitled “Immune response to PCV2 vaccination in PRRSV viremic piglets” was aimed to evaluate whether PRRSV could interfere with the activation of the immune response to PCV2 vaccination in pigs. In this trial, 200 pigs were divided into 2 groups: PCV2-vaccinated (at 4 weeks of age) and PCV2-unvaccinated (control group). Some piglets of both groups got infected by PRRSV, as determined by PRRSV viremia detection, so that 4 groups were defined as follows: PCV2 vaccinated - PRRSV viremic PCV2 vaccinated - PRRSV non viremic PCV2 unvaccinated - PRRSV viremic PCV2 unvaccinated - PRRSV non viremic The following parameters were evaluated in the 4 groups: number of PCV2-specific IFN-γ secreting cells, antibody titres by ELISA and IPMA. Based on the immunological data analysis, it can be deduced that: 1) The low levels of antibodies against PCV2 in the PCV2-vaccinated – PRRSV-viremic group at vaccination (4 weeks of age) could be related to a reduced colostrum intake influenced by PRRSV viremia. 2) Independently of the viremia status, serological data of the PCV2-vaccinated group by ELISA and IPMA does not show statistically different differences. Consequently, it can be be stated that, under the conditions of the study, PRRSV does not interfere with the antibody response induced by the PCV2 vaccine. 3) The cell-mediated immune response in terms of number of PCV2-specific IFN-γ secreting cells in the PCV2-vaccinated – PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN-γ secreting cells after PCV2 vaccination, compared to the PCV2-vaccinated – PRRSV-non-viremic group. The data highlight and further support the inhibitory role of PRRSV on the development and activation of the immune response and highlight how a natural infection at early age can negatively influence the immune response to other pathogens/antigens. The third study entitled “Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8αβ+ T cell subsets by an antibody-derived killer peptide (KP)” was aimed to determine whether and how the killer peptide (KP) could modulate the immune response in terms of activation of specific lymphocyte subsets. This is a preliminary approach also aimed to subsequently evaluate such KP with a potential antivural role or as adjuvant. In this work, pig peripheral blood mononuclear cells (PBMC) were stimulated with three KP concentrations (10, 20 and 40 g/ml) for three time points (24, 48 and 72 hours). TIME POINTS (hours) KP CONCENTRATIONS (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 By using flow cytometry, the qualitative and quantitative modulation of the following immune subsets was evaluated upon KP stimulation: monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and CD4+ and CD8α/β+ T lymphocyte subsets. Based on the data, it can be deduced that: 1) KP promotes a dose-dependent activation of monocytes, particularly after 24 hours of stimulation, by inducing a monocyte phenotypic and maturation shift mainly involved in sustaining the innate/inflammatory response. 2) KP induces a strong dose-dependent modulation of NK and NKT cells, characterized by an intense increase of the NKT cell fraction compared to NK cells, both subsets involved in the antibody-dependent cell cytotoxicity (ADCC). The increase is observed especially after 24 hours of stimulation. 3) KP promotes a significant activation of the cytotoxic T lymphocyte subset (CTL). 4) KP can modulate both the T helper and T cytotoxic phenotype, by inducing T helper cells to acquire the CD8α thus becoming doube positive cells (CD4+CD8+) and by inducing CTL (CD4-CD8+high) to acquire the double positive phenotype (CD4+CD8α+high). Therefore, KP may induce several effects on different immune cell subsets. For this reason, further research is needed aimed at characterizing each “effect” of KP and thus identifying the best use of the decapeptide for vaccination practice, therapeutic purposes or as vaccine adjuvant. RIASSUNTO Il virus della PRRS (Porcine Reproductive Respiratory Syndrome) è uno dei più diffusi agenti patogeni negli allevamenti suini di tutto il mondo, responsabile di una sindrome riproduttiva e respiratoria causa di gravi danni ad impatto sanitario ed economico. Questo virus è emerso attorno alla fine degli anni ’80 ma nonostante siano passati circa una trentina di anni, le conoscenze su alcuni punti essenziali che riguardano le caratteristiche del virus (patogenesi, risposta immunitaria, epidemiologia) appaiono ancora spesso incomplete. Considerando che lo sviluppo dei vaccini moderni è basato sui principi dell’immunità innata e acquisita è essenziale una sempre più completa conoscenza del sistema immunitario inteso come modulazione/regolazione molecolare della risposta infiammatoria e immunitaria in corso di tale infezione. Questo lavoro di tesi, suddiviso in tre diversi studi, ha l’intento di contribuire all’aumento delle informazioni riguardo l’interazione del sistema immunitario, con il virus della PRRS in condizioni di infezione naturale. L’obbiettivo del primo studio, intitolato “Associazione di cellule memoria, cellule citotossiche e cellule secernenti IFN- nella risposta immunitaria in corso di infezione naturale da Virus della Sindrome Riproduttiva e Respiratoria del Suino (PRRSV)” è stato di valutare l’attivazione e la modulazione della risposta immunitaria in suini naturalmente infetti da PRRSV rispetto ad un gruppo controllo non infetto. I parametri valutati sono stati la viremia mediante PCR, il titolo anticorpale mediante ELISA, il numero di cellule secernenti IFN- (IFN- SC) mediante tecnica ELISPOT e la fenotipizzazione di alcune sottopopolazioni linfocitarie (Cellule citotossiche, linfociti T memoria e linfociti T citotossici) mediante citofluorimetria a flusso. Dai risultati ottenuti è stato possibile osservare che l’attivazione della risposta immunitaria cellulo-mediata verso PRRSV appare ritardata durante l’infezione e che l’andamento, in termini di IFN- SC e dei cambiamenti delle sottopopolazioni linfocitarie, mostra comunque degli incrementi seppur successivi nel tempo. E’ stato inoltre osservato che gli andamenti delle diverse sottopopolazioni immunitarie cellulari appaiono temporalmente associati ai livelli di IFN- SC PRRSV-specifiche e ciò potrebbe essere interpretato sulla base del ruolo funzionale che tali sottopopolazioni linfocitarie potrebbero avere nella produzione/secrezione specifica della citochina immunoattivatrice IFN-. Questi dati inoltre supportano l’ipotesi che l’età degli animali alla comparsa dell’infezione o, come tipicamente ipotizzato nell’infezione da PRRSV, le differenti caratteristiche immunobiologiche dell’isolato di campo, sia condizioni critiche nell’ influenzare l’andamento qualitativo e quantitativo della risposta cellulo-mediata durante l’infezione naturale da PRRSV. Il secondo studio, dal titolo “Valutazione della risposta immunitaria nei confronti di una vaccinazione contro PCV2 in suini riscontrati PRRSV viremici e non viremici alla vaccinazione” ha avuto lo scopo di valutare se il virus della PRRS potesse andare ad interferire sull’attivazione della risposta immunitaria indotta da vaccinazione contro PCV2 nel suino. In questo lavoro sono stati arruolati 200 animali divisi in due gruppi, PCV2 Vaccinato (a 4 settimane di età) e PCV2 Non Vaccinato (controllo negativo). Alcuni suinetti di entrambi i gruppi, si sono naturalmente infettati con PRRSV, come determinato con l’analisi della viremia da PRRSV, per cui è stato possibile creare quattro sottogruppi, rispettivamente: PCV2 vaccinato - PRRSV viremico PCV2 vaccinato - PRRSV non viremico PCV2 non vaccinato - PRRSV viremico PCV2 non vaccinato - PRRSV non viremico Su questi quattro sottogruppi sono stati valutati i seguenti parametri: numero di cellule secernenti IFN- PCV2 specifiche, ed i titoli anticorpali mediante tecniche ELISA ed IPMA. Dall’analisi dei dati immunologici derivati dalle suddette tecniche è stato possibile dedurre che:  I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico già al periodo della vaccinazione (4 settimane di età) potrebbero essere messi in relazione ad una ridotta assunzione di colostro legata allo stato di viremia da PRRSV  Indipendentemente dallo stato viremico, i dati sierologici del gruppo vaccinato PCV2 provenienti sia da ELISA sia da IPMA non mostrano differenze statisticamente significative. Di conseguenza è possibile affermare che in questo caso PRRSV non interferisce con la risposta anticorpale promossa dal vaccino PCV2.  La risposta immunitaria cellulo-mediata, intesa come numero di cellule secernenti IFN- PCV2 specifiche nel gruppo PCV2 vaccinato PRRS viremico sembra essere compromessa, come viene infatti dimostrato dalla diminuzione del numero di cellule secernenti IFN- dopo la vaccinazione contro PCV2, comparata con il gruppo PCV2 vaccinato- non viremico. I dati evidenziano ed ulteriormente sostengono il ruolo inibitorio del virus della PRRSV sullo sviluppo ed attivazione della risposta immunitaria e come un infezione naturale ad età precoci possa influenzare negativamente la risposta immunitaria ad altri patogeni/antigeni. Il terzo studio, intitolato “Modulazione fenotipica di: monociti CD14+, cellule natural killer (NK), T natural killer (NKT) e sottopopolazioni linfocitarie T CD4+ e CD8+ durante stimolazione con killer peptide (KP) nella specie suina” ha avuto come scopo quello di stabilire se e come il Peptide Killer (KP) potesse modulare la risposta immunitaria in termini di attivazione di specifiche sottopopolazioni linfocitarie. Si tratta di un approccio preliminare anche ai fini di successivamente valutare tale KP in un potenziale ruolo antivirale o come adiuvante. In questo lavoro, periferal blood mononuclear cells (PBMC) suine sono state stimolate con KP a tre diverse concentrazioni (10, 20 e 40 g/ml) per tre diversi tempi (24, 48 e 72 ore). TEMPI DI STIMOLAZIONE (ore) CONCENTRAZIONE DI KP (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 Mediante la citometria a flusso è stato dunque possibile analizzare il comportamento qualitativo e quantitativo di alcune sottopopolazioni linfocitarie sotto lo stimolo del KP, tra cui: monociti, cellule Natural Killer (NK), cellule T Natural Killer (NKT) e linfociti T CD4 e CD8+. Dai dati ottenuti è stato possibile dedurre che: 1) KP promuove un’attivazione dei monociti dose-dipendente in particolare dopo 24 ore di stimolazione, inducendo uno “shift” fenotipico e di maturazione monocitaria maggiormente coinvolto nel sostegno della risposta innata/infiammatoria. 2) KP induce una forte modulazione dose-dipendente di cellule NK e NKT con un forte aumento della frazione delle cellule NKT rispetto alle NK, sottopopolazioni entrambe coinvolte nella citotossicità cellulare mediata da anticorpi (ADCC). L’aumento è riscontrabile soprattutto dopo 24 ore di stimolazione. 3) KP promuove una significativa attivazione della sottopopolazione del linfociti T citotossici (CTL). 4) Per quanto riguarda la marcatura CD4+/CD8+ è stato dimostrato che KP ha la capacità di modulare sia il fenotipo T helper che T citotossico, inducendo le cellule T helper ad acquisire CD8 diventando quindi doppio positive (CD4+CD8+) ed inducendo il fenotipo CTL (CD4-CD8+high) ad acquisire il fenotipo doppio positivo (CD4+CD8α+high). Molti dunque potrebbero essere gli effetti che il decapeptide KP potrebbe esercitare sulle diverse sottopopolazioni del sistema immunitario, per questo motivo va evidenziata la necessità di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna “abilità” di KP e che conducano successivamente alla scoperta del migliore utilizzo che si possa fare del decapeptide sia dal punto di vista vaccinale, terapeutico oppure sotto forma di adiuvante vaccinale.

PLGA microspheres for the delivery of a novel subunit TB vaccine

Biodegradable poly(dl-lactide-co-glycolide) microspheres were prepared using a modified double emulsion solvent evaporation method for the delivery of the subunit tuberculosis vaccine (Ag85B-ESAT-6), a fusion protein of the immunodominant antigens 6-kDa early secretory antigenic target (ESAT-6) and antigen 85B (Ag85B). Addition of the cationic lipid dimethyl dioctadecylammonium bromide (DDA) and the immunostimulatory trehalose 6,6'-dibehenate (TDB), either separately or in combination, was investigated for the effect on particle size and distribution, antigen entrapment efficiency, in vitro release profiles and in vivo performance. Optimised formulation parameters yielded microspheres within the desired sub-10 mu m range (1.50 +/- 0.13 mu m), whilst exhibiting a high antigen entrapment efficiency (95 +/- 1.2%) and prolonged release profiles. Although the microsphere formulations induced a cell-mediated immune response and raised specific antibodies after immunisation, this was inferior to the levels achieved with liposomes composed of the same adjuvants (DDA-TDB), demonstrating that liposomes are more effective vaccine delivery systems compared with microspheres.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′- dibehenate) - A novel adjuvant inducing both strong CMI and antibody responses:a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4°C and 25°C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

MC1R-dependent, melanin-based colour polymorphism is associated with cell-mediated response in the Eleonora's falcon.

Colour polymorphism in vertebrates is usually under genetic control and may be associated with variation in physiological traits. The melanocortin 1 receptor (Mc1r) has been involved repeatedly in melanin-based pigmentation but it was thought to have few other physiological effects. However, recent pharmacological studies suggest that MC1R could regulate the aspects of immunity. We investigated whether variation at Mc1r underpins plumage colouration in the Eleonora's falcon. We also examined whether nestlings of the different morphs differed in their inflammatory response induced by phytohemagglutinin (PHA). Variation in colouration was due to a deletion of four amino acids at the Mc1r gene. Cellular immune response was morph specific. In males, but not in females, dark nestling mounted a lower PHA response than pale ones. Although correlative, our results raise the neglected possibility that MC1R has pleiotropic effects, suggesting a potential role of immune capacity and pathogen pressure on the maintenance of colour polymorphism in this species.

CELL-MEDIATED AND HUMORAL IMMUNE-RESPONSES IN IMMUNIZED AND OR DERMATOBIA-HOMINIS INFESTED RABBITS

The cell-mediated and humoral immune response of rabbits to antigens from larvae of Dermatobia hominis were analyzed by leucocyte migration inhibition factor assay (MIF), immunodiffusion (ID) and passive hemagglutination (PH) test in rabbits immunized with D. hominis extract, in rabbits immunized and infested with the parasite and rabbits infested with D. hominis. Twenty rabbits were divided into five groups: Group 1, rabbits immunized with a crude antigen extract, evaluated for 40 weeks at 4 week intervals; Group 2, rabbits immunized and infested with newly hatched larvae at 14 weeks post immunization (PI) and evaluated as Group 1; Group 3, rabbits immunized, evaluated for 28 weeks at 2 week intervals; Group 4, rabbits immunized and infested at 4 weeks PI and evaluated as Group 3; Group 5, rabbits infested and evaluated for 24 weeks at 2 week intervals. Different patterns of reactivity were observed in the infested and immunized animals: immunized rabbits developed antibodies and cellular immune responses earlier and at higher levels during immunization than the infested rabbits; the infestation at 14 weeks PI, when the cell-mediated and humoral immune response began to decrease, or at 4 weeks PI when these parameters were at higher levels, elicited an anamnestic response. After the spontaneous elimination of larvae by the host, from the 4th week PI onwards, high titers of antibodies and migration inhibition indices were maintained for a long period. These results suggest that the onset of cellular and humoral immune responses after immunization may be important as a biological control of myiasis and contribute to better understanding of the immune defense mechanism of the host against D. hominis.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Developing solid particulate vaccine adjuvants:surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease

Recent studies have demonstrated that angiogenesis and suppressed cell- mediated immunity (CMI) play a central role in the pathogenesis of malignant disease facilitating tumour growth, invasion and metastasis. In the majority of tumours, the malignant process is preceded by a pathological condition or exposure to an irritant which itself is associated with the induction of angiogenesis and/or suppressed CMI. These include: cigarette smoking, chronic bronchitis and lung cancer; chronic oesophagitis and oesophageal cancer; chronic viral infections such as human papilloma virus and ano-genital cancers, chronic hepatitis B and C and hepatocellular carcinoma, and Epstein- Barr virus (EBV) and lymphomas; chronic inflammatory conditions such as Crohn's disease and ulcerative colitis and colorectal cancer; asbestos exposure and mesothelioma and excessive sunlight exposure/sunburn and malignant melanoma. Chronic exposure to growth factors (insulin-like growth factor-I in acromegaly), mutations in tumour suppressor genes (TP53 in Li Fraumeni syndrome) and long-term exposure to immunosuppressive agents (cyclosporin A) may also give rise to similar environments and are associated with the development of a range of solid tumours. The increased blood supply would facilitate the development and proliferation of an abnormal clone or clones of cells arising as the result of: (a) an inherited genetic abnormality; and/or (b) acquired somatic mutations, the latter due to local production and/or enhanced delivery of carcinogens and mutagenic growth factors. With progressive detrimental mutations and growth-induced tumour hypoxia, the transformed cell, to a lesser or greater extent, may amplify the angiogenic process and CMI suppression, thereby facilitating further tumour growth and metastasis. There is accumulating evidence that long-term treatment with cyclo-oxygenase inhibitors (aspirin and indomethacin), cytokines such as interferon-α, anti-oestrogens (tamoxifen and raloxifene) and captopril significantly reduces the incidence of solid tumours such as breast and colorectal cancer. These agents are anti-angiogenic and, in the case of aspirin, indomethacin and interferon-α have proven immunomodulatory effects. Collectively these observations indicate that angiogenesis and suppressed CMI play a central role in the development and progression of malignant disease. (C) 2000 Elsevier Science Ltd.