Role of B cells in antigen presentation of the hepatitis B���core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure���function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

Immune response to a differentiation antigen induced by altered antigen: A study of tumor rejection and���autoimmunity

Recognition of self is emerging as a theme for the immune recognition of human cancer. One question is whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells. A second but related question is whether immune recognition of tissue autoantigens can actually induce tumor rejection. To address these issues, a mouse model was developed to investigate immune responses to a melanocyte differentiation antigen, tyrosinase-related protein 1 (or gp75), which is the product of the brown locus. In mice, immunization with purified syngeneic gp75 or syngeneic cells expressing gp75 failed to elicit antibody or cytotoxic T-cell responses to gp75, even when different immune adjuvants and cytokines were included. However, immunization with altered sources of gp75 antigen, in the form of either syngeneic gp75 expressed in insect cells or human gp75, elicited autoantibodies to gp75. Immunized mice rejected metastatic melanomas and developed patchy depigmentation in their coats. These studies support a model of tolerance maintained to a melanocyte differentiation antigen where tolerance can be broken by presenting sources of altered antigen (e.g., homologous xenogeneic protein or protein expressed in insect cells). Immune responses induced with these sources of altered antigen reacted with various processed forms of native, syngeneic protein and could induce both tumor rejection and autoimmunity.

Abolition of morphine-immunosuppression in mice lacking the ��-opioid receptor gene

Opiates are potent analgesic and addictive compounds. They also act on immune responses, and morphine, the prototypic opiate, has been repeatedly described as an immunosuppressive drug. Pharmacological studies have suggested that the inhibitory action of opiates on immunity is mediated by multiple opioid receptor sites but molecular evidence has remained elusive. Recently, three genes encoding ��- (MOR), ��-, and ��-opioid receptors have been cloned. To investigate whether the ��-opioid receptor is functionally implicated in morphine immunosuppression in vivo, we have examined immune responses of mice with a genetic disruption of the MOR gene. In the absence of drug, there was no difference between wild-type and mutant mice with regard to a large number of immunological endpoints, suggesting that the lack of MOR-encoded protein has little consequence on immune status. Chronic morphine administration induced lymphoid organ atrophy, diminished the ratio of CD4+CD8+ cells in the thymus and strongly reduced natural killer activity in wild-type mice. None of these effects was observed in MOR-deficient mice after morphine treatment. This demonstrates that the MOR gene product represents a major molecular target for morphine action on the immune system. Because our previous studies of MOR-deficient mice have shown that this receptor protein is also responsible for morphine analgesia, reward, and physical dependence, the present results imply that MOR-targeted therapeutic drugs that are developed for the treatment of pain or opiate addiction may concomitantly influence immune responses.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Implications of immune-to-brain communication for sickness and pain

This review presents a view of hyperalgesia and allodynia not typical of the field as a whole. That is, exaggerated pain is presented as one of many natural consequences of peripheral infection and injury. The constellation of changes that results from such immune challenges is called the sickness response. This sickness response results from immune-to-brain communication initiated by proinflammatory cytokines released by activated immune cells. In response to signals it receives from the immune system, the brain orchestrates the broad array of physiological, behavioral, and hormonal changes that comprise the sickness response. The neurocircuitry and neurochemistry of sickness-induced hyperalgesia are described. One focus of this discussion is on the evidence that spinal cord microglia and astrocytes are key mediators of sickness-induced hyperalgesia. Last, evidence is presented that hyperalgesia and allodynia also result from direct immune activation, rather than neural activation, of these same spinal cord glia. Such glial activation is induced by viruses such as HIV-1 that are known to invade the central nervous system. Implications of exaggerated pain states created by peripheral and central immune activation are discussed.

Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?

Painful peripheral neuropathies are precipitated by nerve injury from disease or trauma. All such injuries will be accompanied by an inflammatory reaction, a neuritis, that will mobilize the immune system. The role of the inflammation itself is difficult to determine in the presence of structural damage to the nerve. A method has been devised to produce a focal neuritis in the rat sciatic nerve that involves no more than trivial structural damage to the nerve. This experimental focal neuritis produces neuropathic pain sensations (heat- and mechano-hyperalgesia, and cold- and mechano-allodynia) in the ipsilateral hind paw. The abnormal pain sensations begin in 1���2 days and last for 4���6 days, with a subsequent return to normal. These results suggest that there is a neuroimmune interaction that occurs at the outset of nerve injury (and perhaps episodically over time in slow developing conditions like diabetic neuropathy) that produces neuropathic pain. The short duration of the phenomena suggest that they may prime the system for more slowly developing mechanisms of abnormal pain (e.g., ectopic discharge in axotomized primary afferent neurons) that underlie the chronic phase of painful neuropathy.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ �� DW/J)F1 background. Mutant dwJ/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhrlit mutation, which like the Pit1dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1dw mutant, and the closely related, long-lived Prop-1df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Toward a new generation of vaccines: The anti-cytokine therapeutic vaccines

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector���target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.

Development and characterization of a rodent model of immune-mediated cholangitis.

The cholangiopathies are a group of hepatobiliary diseases in which intrahepatic bile duct epithelial cells, or cholangiocytes, are the target for a variety of destructive processes, including immune-mediated damage. We tested the hypothesis that cholangitis could be induced in rodents by immunization with highly purified cholangiocytes. Inbred Wistar rats were immunized with purified hyperplastic cholangiocytes isolated after bile duct ligation from either syngeneic Wistar or allogeneic Fischer 344 rats; control rats were immunized with bovine serum albumin (BSA) or hepatocytes. After immunization with cholangiocytes, recipient animals developed histologic evidence of nonsuppurative cholangitis without inflammation in other organs; groups immunized with BSA or hepatocytes showed no cholangitis. Immunohistochemical studies revealed that portal tract infiltrates around bile ducts consisted of CD3-positive lymphocytes, some of which expressed major histocompatibility complex class II antigen; B cells and exogenous monocytes/macrophages were essentially absent. Transfer of unfractionated ConA-stimulated spleen cells from cholangiocyte-immunized (but not BSA-immunized) rats into recipients also caused nonsuppurative cholangitis. Moreover, these splenocytes from cholangiocyte-immunized (but not BSA-immunized) rats were cytotoxic in vitro for cultured rodent cholangiocytes; no cytotoxicity was observed against a rat hepatocyte cell line. Also, a specific antibody response in sera of cholangiocyte-immunized rats was demonstrated by immunoblots against cholangiocyte proteins. Finally, cholangiograms in cholangiocyte-immunized rats showed distortion and tortuosity of the entire intrahepatic biliary ductal system. This unique rodent model of experimental cholangitis demonstrates the importance of immune mechanisms in the pathogenesis of cholangitis and will prove useful in exploring the mechanisms by which the immune system targets and damages cholangiocytes.

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency.

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

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-�� 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-�� 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-�� 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-�� 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-��. 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-�� 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-�� secreting cells in the PCV2-vaccinated ��� PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN-�� 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 CD8����+ 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 CD8��/��+ 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 CD8�� thus becoming doube positive cells (CD4+CD8���+) and by inducing CTL (CD4-CD8���+high) to acquire the double positive phenotype (CD4+CD8��+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) �� uno dei pi�� 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 �� 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 �� basato sui principi dell���immunit�� innata e acquisita �� essenziale una sempre pi�� 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)��� �� 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 �� 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 ci�� 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���et�� 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 et��) 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 �� 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 �� stato possibile dedurre che: ��� I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico gi�� al periodo della vaccinazione (4 settimane di et��) 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 �� 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 et�� 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 �� 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 �� 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 citotossicit�� cellulare mediata da anticorpi (ADCC). L���aumento �� 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���+ �� stato dimostrato che KP ha la capacit�� 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+CD8��+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 necessit�� di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna ���abilit����� 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.

Influence of different genotypes in the pattern of selenoprotein expression in response to Brazil nut supplementation

The micronutrient selenium is essential to human physiology. As the amino acid selenocysteine, it is inserted into selenoproteins with a wide range of functions including antioxidant capacity, thyroid hormone metabolism, improvement of immune system, brain function, fertility and reproduction. Low selenium status has been associated with increased risk for chronic diseases, such as cancer, type-2 diabetes and cardiovascular disease. In this context, several studies have been conducted in order to investigate if selenium supplementation could reduce the risk of such diseases. However, genetic variations may interfere in the response of individuals to a dietary intervention and must be considered as a important source of inter-individual variation. Therefore, this study was conducted was conducted to investigate the influence of genetic variations in selenoproteins genes on the response to an intervention with Brazil nuts, the richest source of selenium known in nature. The study included 130 healthy volunteers with both genders, aged 20 to 60 years old selected in University of S��o Paulo. They received nuts for 8 weeks, eating one nut a day, and did a washout period for more 8 weeks. All volunteers had a blood sampling collection every 4 weeks during 4 months, in a total of 5. The following analysis were done: anthropometric measurements, lipid profile, plasma malondialdehyde, plasma and erythrocyte Se, selenoprotein P, plasma and erythrocyte GPx activity, gene expression of GPX1, SEPP1, SELS and SEP15. The volunteers were also genotyped for SNPs rs1050450, rs3811699, rs1800699, rs713041, rs3877899, rs7579, rs34713741 and rs5845. Each unit of Brazil nut provided an average of 300 µg of selenium. All 130 volunteers completed the protocol. The concentrations of total cholesterol and glucose decreased after 8 weeks of supplementation. Moreover, HDL concentrations were higher for carriers of the variant T allele for GPX4_rs713041. The frequencies of the variant genotypes were 5,4% for rs1050450, rs3811699 e rs1088668, 10% for rs3877899, 19,2% for rs713041 e rs7579, 11,5% for rs5845 and 8,5% for rs34713741. The levels of the five biomarkers increased significantly after supplementation. In addition, erythrocyte GPx activity was influenced by rs1050450, rs713041 and rs5845; erythrocyte selenium was influenced by rs5845 and plasma selenium by rs3877899. Gene expression of GPX1, SEPP1 and SEP15 were higher after supplementation. The SNP rs1050450 influenced GPX1 mRNA expression and rs7579 influenced SEPP1 mRNA expression. Therefore, it can be concluded that the supplementation with one of Brazil nut for 8 weeks was efficient to reduce total cholesterol and glucose levels and to increase the concentrations of the main biomarkers of selenium status in healthy adults. Furthermore, our results suggest that GPX4_rs713041 might interfere on HDL concentrations and GPx1 activity, GPX1_rs1050450 might interfere on GPx1 activity, SEP15_rs5845 might interfere on GPx1 activity and erythrocyte selenium and SEPP1_3877899 might interfere on plasma Se levels. Therefore, the effect of genetic variations should be considered in future nutritional interventions evaluating the response to Brazil nut supplementation.

Efeito da suplementa����o prolongada de gr��o de soja cru e integral no pr��-parto sobre o desempenho produtivo, qualidade oocit��ria e embrion��ria, e fun����o imune de vacas leiteiras

Objetivou-se avaliar o efeito da suplementa����o prolongada de gr��o de soja cru e integral (GSI) como fonte de ��cido graxo &#937;6 sobre o desempenho produtivo, perfil metab��lico, qualidade oocit��ria e embrion��ria e fun����o imune de vacas leiteiras no per��odo de transi����o e in��cio de lacta����o. Foram selecionadas 44 vacas da ra��a Holandesa, mult��paras e gestantes, com parto previsto para 90 dias ap��s o in��cio da avalia����o e fornecimento das dietas experimentais, por��m em raz��o da ocorr��ncia de enfermidades metab��licas ou infecciosas (3 abortos; 3 deslocamentos de abomaso; 3 enfermidades podais; 4 distocias) 13 animais foram retirados do experimento. As vacas foram distribu��das aleatoriamente em quatro grupos experimentais diferindo entre eles o in��cio do fornecimento de gr��o de soja cru e integral (GSI) durante o pr��-parto. A dieta era baseada na inclus��o de 12% de GSI %MS, com aproximadamente 5,1% de extrato et��reo (EE) o in��cio de seu fornecimento foi conforme descrito a seguir: Grupo 0: Animais n��o receberam dieta contendo GSI no pr��-parto; Grupo 30: In��cio do fornecimento de dieta com GSI nos 30 dias finais da gesta����o; Grupo 60: In��cio do fornecimento de dieta com GSI nos 60 dias finais da gesta����o; Grupo 90: In��cio do fornecimento de dieta com GSI nos 90 dias finais da gesta����o. Ap��s o parto, todas as vacas receberam dieta ��nica com 5,1% de EE, baseada na inclus��o de 12% de GSI %MS at�� 90 dias de lacta����o. Os animais foram arra��oados de acordo com o consumo de mat��ria seca no dia anterior, de forma a ser mantido porcentual de sobras das dietas, diariamente, entre 5 e 10%. As amostras dos alimentos e sobras foram coletadas diariamente e armazenadas a -20��C. Semanalmente as amostras coletadas diariamente foram misturadas e foi retirada uma amostra composta referente a um per��odo de uma semana, a fim de mensurar o consumo de mat��ria seca e nutrientes. Amostras de fezes foram coletadas nos dias -56, -21, 21, 56 e 84 dias em rela����o ao parto, com o prop��sito de mensurar a digestibilidade da mat��ria seca e nutrientes. A produ����o de leite foi mensurada diariamente e para a composi����o dos teores de gordura, prote��na, lactose e perfil de ��cidos graxos amostras foram coletadas semanalmente. As amostras de sangue para an��lise dos metab��litos sangu��neos foram coletadas semanalmente. Amostras de sangue para mensurar a atividade do sistema imune foram coletadas na semanas -8, -4, -2, -1 em rela����o ao parto, parto, +1, +2, +4 e +8 semanas no per��odo p��s-parto. Nos dias 21, 42, 63 e 84 do per��odo p��s-parto foram realizadas aspira����es foliculares, com posterior fertiliza����o in vitro dos o��citos. Todas as vari��veis mensuradas foram analisadas pelo procedimento PROC MIXED do SAS 9.4 atrav��s de regress��o polinomial, utilizando efeito fixo de tratamento, semana, intera����o tratamento*semana e efeito de animal dentro de tratamento como aleat��rio. Utilizou n��vel de 5% de signific��ncia. Foi observado efeito (P<0,05) linear crescente para CEE no pr��-parto. N��o foi observado diferen��as no CMS e nutrientes no p��s-parto. N��o houve altera����o da digestibilidade nos per��odos pr�� e p��s-parto. N��o houve altera����o no balan��o de energia e nitrog��nio nos periodos pr�� e p��s-parto. N��o foi observado diferen��a na produ����o, composi����o e teor dos componentes totais do leite. No perfil de ��cidos graxos do leite houve efeito (P<0,05) linear descrescente para as concentra����es de C16:1cis, C18:1 cis, total de C:18 insaturado, total de AG monoinsaturados, insaturados e a rela����o do total de AGS:AGI. Foi observado efeito linear (P<0,05) crescente para o total de AG aturado e efeito (P<0,05) quadr��tico para C18:2, CLAcis9-trans11, e total de AGPI. Foi observado efeito linear crescente (P<0,05) para colesterol total, LDL no pr��parto e linear decrescente (P<0,05) para GGT nos per��odos pr�� e p��s-parto. Foi observado efeito quadr��tico (P<0,05) para HDL no pr��-parto e AST no p��s-parto. Em rela����o a atividade do sistema imune foi observado efeito linear (P<0,05) crescente para o percentual de CD3+ ativos no p��s-parto, para o percentual de mon��citos que produziram esp��cie reativa de oxig��nio (ERO) no p��s-parto quando foram estimulados por S.aureus e E.coli e para a intensidade de imunofluoresc��ncia de ERO para ganul��citos no p��s-parto quando estimulados por S.aureus. Foi observado efeito (P<0,05) quadr��tico para o percentual de granul��citos, mononucleares, CD8+ ativos no p��s-parto e para o percentual de granul��citos que produziram ERO no p��s-parto quando estimulados por E.coli. A suplementa����o prolongada com GSI no pr��-parto melhora a atividade do sistema imune, n��o melhora a qualidade oocit��ria e embrion��ria bem como n��o influencia negativamente os parametros produtivos de vacas leiteiras no per��odo de transi����o e in��cio de lacta����o

An��lise fenot��pica de c��lulas dendr��ticas e de linf��citos T efetores, polifuncionais e reguladores no l��quen plano

INTRODU����O: L��quen plano (LP) �� uma doen��a mucocut��nea de natureza inflamat��ria cr��nica de etiologia ainda desconhecida. A estimula����o da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as c��lulas dendr��ticas e direcionar a resposta de c��lulas T CD4+ e CD8+ efetoras, assim como tamb��m favorecer o estado inflamat��rio do LP. OBJETIVOS: Avaliar o perfil fenot��pico de c��lulas dendr��ticas miel��ides (mDCs) e plasmocit��ides (pDCs) e de linf��citos T CD4+ e CD8+ ap��s est��mulo com agonistas de TLRs no sangue perif��rico de pacientes com LP. Al��m disto, avaliar a frequ��ncia, perfil de matura����o e os subtipos de c��lulas T CD4+ e TCD8+ reguladores. M��TODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 �� 4,73 anos de idade e um grupo controle com 22 indiv��duos sadios (18 mulheres, 4 homens), com 43,92 �� 7,83 anos de idade. As c��lulas mononucleares (CMNs) de sangue perif��rico foram avaliadas por citometria de fluxo quanto ��: 1) Produ����o de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) An��lise de c��lulas T CD4+ e CD8+ monofuncionais e polifuncionais ap��s est��mulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avalia����o de c��lulas Th17 e Th22/Tc22 em CMNs ap��s est��mulo com SEB; 4) Frequ��ncia, perfil de matura����o e subtipos de c��lulas T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequ��ncia de mDCs TNF-alfa+ ap��s est��mulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminui����o da express��o de CD83. J�� nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a express��o de CD80 e o CpG/TLR9 diminuiu a express��o de CD83 no LP. 2) As c��lulas T CD4+ secretoras de IL-10 mostraram aumento da frequ��ncia nos n��veis basais, que diminuiu ap��s est��mulo com LPS e SEB. Em contraste, a produ����o de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As c��lulas T CD4+ polifuncionais, secretoras de 5 citocinas simult��neas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminu��ram no LP ap��s est��mulo com CL097 e CpG. Entretanto, na aus��ncia de IL-10, houve aumento da frequ��ncia de c��lulas CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em c��lulas TCD4+ que expressam CD38, marcador de ativa����o cr��nica e na aus��ncia de IL-10. Similarmente, ��s TCD4+, uma diminui����o de c��lulas T CD8+ IFN-y+ e TNF+ foram detectadas ap��s est��mulo com CpG. 3) As c��lulas Th22/Tc22 nos n��veis basais e ap��s est��mulo com SEB se mostraram aumentadas. As c��lulas Th17 n��o mostraram diferen��as entre os grupos. 4) A frequ��ncia das c��lulas T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) est�� elevada no LP. Quanto aos perfis de matura����o, h�� aumento na frequ��ncia de c��lulas TCD4+ de mem��ria efetora enquanto que para as c��lulas T CD8+ h�� predom��nio das c��lulas de mem��ria central. Quanto aos subtipos, h�� aumento nas c��lulas T CD4+ regs perif��ricas (pT reg). CONCLUS��ES: O estado de ativa����o das mDCs ap��s ativa����o das vias de TLRs 4 e 7/8 pode influenciar na gera����o de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos est��mulos TLRs reflete a ativa����o destas c��lulas no sangue perif��rico. Al��m disso, o aumento de Th22/Tc22 e das c��lulas T regs indicam uma rela����o entre regula����o e c��lulas efetoras no sangue perif��rico evidenciando que existem altera����es extracut��neas no LP