Different degrees of malnutrition and immunological alterations according to the aetiology of cirrhosis: a prospective and sequential study

Abstract Objectives In this work we investigated how immunological dysfunction and malnutrition interact in alcoholic and viral aetiologies of cirrhosis. Methods To investigate the matter, 77 cirrhotic patients divided in three aetiologies [Alcohol, HCV and Alcohol + HCV) and 32 controls were prospectivelly and sequentially studied. Parameters of humoral immunity (Components 3 and 4 of seric complement and immunoglobulins A M, G and E) and of cellular immunity (total leukocytes and lymphocytes in peripheral blood, T lymphocytes subpopulations, CD4+ and CD8+, CD4+/CD8+ ratio and intradermic tests of delayed hypersensitivity), as well as nutrititional parameters: anthropometric measures, serum albumin and transferrin were evaluated. Results Multiple statistical comparisons showed that IgM was higher in HCV group; IgG was significantly elevated in both HCV and Alcohol + HCV, whereas for the Alcohol group, IgE was found at higher titles. The analysis of T- lymphocytes subpopulations showed no aetiologic differences, but intradermic tests of delayed hypersensitivity did show greater frequency of anergy in the Alcohol group. For anthropometric parameters, the Alcohol +HCV group displayed the lowest triceps skinfold whereas creatinine ��� height index evaluation was more preserved in the HCV group. Body mass index, arm muscle area and arm fat area showed that differently from alcohol group, the HCV group was similar to control. Conclusion Significant differences were found among the main aetiologies of cirrhosis concerning immunological alterations and nutritional status: better nutrition and worse immunology for HCV and vice-versa for alcohol.

Espressione del vascular endothelium growth factor (VEGF) nella micosi fungoide

La micosi fungoide (MF) �� un linfoma a cellule T primitivo della cute usualmente indolente negli stadi iniziali, ma con prognosi decisamente peggiore nelle fasi avanzate, ove attualmente non sono presenti strategie terapeutiche tali da indurre remissioni durature. Recenti osservazioni indicano che alti livelli di espressione del vascular endothelial growth factor (VEGF) nelle cellule della MF sembrano correlare con una prognosi peggiore. Nel presente studio, sono state vagliate le eventuali differenze di espressione di VEGF nella MF e nei linfociti T normali. In primo luogo sono stati raffrontati 63 casi di MF con 20 campioni corrispondenti alle diverse sottopopolazioni di linfociti T normali, per stabilire quale fra questi esprimesse maggiori livelli di VEGF. Tale esperimento ha dimostrato che il gene �� notevolmente pi�� espresso nella MF. Si �� provveduto a stabilire se tale dato sia da correlarsi ad un fenomeno patologico o fisiologico. Quindi sono state eseguite indagini di gene expression profiling (GEP) allo scopo di vagliare i livelli di VEGF nella popolazione linfocitaria T normale (CD4+, CD8+, HLA-DR+ e HLA-DR-): da ci�� �� risultato che i linfociti T attivati esprimono maggiormente VEGF e che il loro GEP �� globalmente paragonabile a quello della MF. Pertanto, i linfociti T attivati sono stati considerati la controparte normale delle cellule della MF. Successivamente si �� confrontata l���espressione quantitativa di VEGF nei linfociti T attivati e nelle cellule della MF, evidenziando come questa sia maggiore nella popolazione neoplastica indipendentemente dallo stadio della malattia. Le indagini immunoistochimiche condotte su 18 casi di MF, hanno confermato quanto evidenziato attraverso il GEP. Concludendo, la ricerca ha dimostrato per la prima volta l���espressione di VEGF negli elementi della MF. Ci�� porta a supporre che la de-regolazione genica della via di VEGF sia correlata nella patogenesi della MF e che tale molecola possa considerarsi un potenziale bersaglio terapeutico.

Misregulation of the tumour suppressor gene CYLD drives hyperactivation of T cells leading to intestinal pathology

The tumour suppressor gene cyld is mutated in familial cylindromatosis, an autosomal-dominant condition that predisposes to multiple skin tumours. The deubiquitinase CYLD acts as a negative regulator of NF-��B signaling. To analyse the function of CYLD in vivo we used the CYLDex7/8 mice, which are characterized by loss of the full-length transcript and overexpression of a short splice variant of CYLD (sCYLD). In CYLDex7/8 mice the overexpression of sCYLD results in splenomegaly and lymphadenopathy. Additionally, the B cell population in spleen and lymph nodes is increased at the expense of T cells. Analysis of CYLDex7/8 T cells showed a significant reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and in the periphery. By investigating the impact of sCYLD in TCR signaling in thymocytes, we could demonstrate that sCYLD partially inhibited the activation of Zap70 and thereby negatively regulated TCR signaling. In vitro as well as in vivo we could show that CD4+ T cells displayed a hyperactive phenotype, proliferated to a better extent than WT cells and expressed high amounts of inflammatory cytokines such as IL-6 and IL-17A. Western Blots of steady state thymocytes and peripheral CD4+ T cells were performed, showing that the noncanonical pathway was highly upregulated visualized by the expression levels of RelB and p100 leading to a hyperactive phenotype of CD4+ T cells. In order to investigate the contribution of sCYLD in positive and negative selection in the thymus in vivo, the HY-TCR transgene (HYtg) was crossed to CYLDex7/8 mice. The analysis of CYLDex7/8 HYtg males revealed an increase in CD4+CD8+ DP as well as in CD8+ SP thymocytes, suggesting a less pronounced negative selection in CYLD mutant mice compared to HYtg control mice. Interestingly, the impaired negative selection in the thymus was accompanied by a strong colitis phenotype at early ages (4 weeks). Since medullary TECs (mTECs) play an important role in the late stage of T cell development by negatively selecting autoreactive thymocytes, the levels of mTECs in the medullary compartment was investigated. Of note, low numbers of mTECs were observed, combined with decreased expression levels of the mTEC markers UEA-1, keratin-5, claudin-3 and claudin-4. The reduction of mTECs in the medullary compartment could explain the inflammatory phenotype of CD4+ T cells in CYLDex7/8 mice leading to the severe intestinal pathology observed in these mice. Taken together, these results show an important role of sCYLD in T cell development and function as well as in NF-��B signaling of T cells.

Erkennung der alpha-Kette des GM-CSF-Rezeptors (CSF2RA) durch einen HLA-unabh��ngigen alpha:beta-T-Zellrezeptor

Aus dem tumorreaktiven T-Zellrepertoire der Melanompatientin Ma-Mel-86/INTH, bei der im Verlauf Lymphknotenmetastasen HLA-Klasse I-negativer Tumorzellen auftraten, wurden durch Stimulation mit autologen Tumorzellen CD8+ T-Zellklone isoliert und expandiert, die auf Melanomzellen der Patientin CSF2RA (engl. GM-CSF receptor alpha chain) in HLA-unabh��ngiger Weise erkannten. Aus einem der T-Zellklone wurde ein CSF2RA-reaktiver ��:��-T-Zellrezeptor (TCR, engl. T-cell receptor) kloniert (Bezeichnung: TCR-1A.3/46). Die ��-Kette des TCR enthielt die Dom��nen TRAV14/DV4*01, TRAJ48*01 und TRAC*01, die ��-Kette die Dom��nen TRBV10-3*01, TRBD2*01, TRBJ2-7*01 und TRBC2*01. Durch Austausch der humanen konstanten gegen die homologen murinen Dom��nen wurde der TCR optimiert (Bezeichnung: cTCR-1A.3/46) und hinsichtlich seiner Expression und Funktionalit��t nach retroviralem Transfer in humane PBMC (engl. peripheral blood mononuclear cells) im 51Chromfreisetzungstest, im IFN-��-ELISpot-Assay und in einem Degranulations-Assay validiert. TCR-transgene T-Zellen lysierten nicht nur spezifisch die HLA-defizienten, CSF2RA+ Melanomlinien des Modells Ma-Mel-86, sondern erkannten auch Zelllinien verschiedener Spezies nach Transfektion von CSF2RA sowie Monozyten, Granulozyten, dendritische Zellen und ein breites Spektrum h��matologischer Malignome myeloiden Ursprungs ungeachtet deren HLA-Ph��notypen. Lymphatische Zellen sowie CD34+ Blutstammzellen wurden in In vitro-Untersuchungen nicht erkannt. Der Zusatz von GM-CSF zu Zellen, die CSF2RA und CSF2RB exprimierten, inhibierte die Erkennung durch TCR-transgene PBMC, w��hrend die Koexpression der ��- und der ��-Kette des GM-CSF-Rezeptors alleine keinen negativen Effekt auf die Erkennung hatte. Daraus war zu schlie��en, dass CSF2RA pr��ferentiell freistehend und weniger nach Integration in den heteromultimerischen GM-CSF-Rezeptor-Komplex erkannt wurde. In der zweidimensionalen Collier-de-Perles-Visualisierung der IMGT-Datenbank (engl. International immunogenetics information system) wies der CSF2RA-reaktive TCR-1A.3/46 im Vergleich zu TCR von konventionellen, HLA-restringierten T-Zellen keine Besonderheiten auf. Dar��ber hinaus waren auch die von den HLA-unabh��ngigen T-Zellen exprimierten CD8-Molek��le identisch zu den CD8-Molek��len HLA-abh��ngiger CTL (engl. cytotoxic T lymphocytes). Die Pr��senz von CD8-Molek��len f��rderte die HLA-unabh��ngige Erkennung von CSF2RA, schien aber daf��r nicht zwingend erforderlich zu sein, da Antik��rper gegen CD8 die Erkennung zu ca. 65 % blockierten und TCR-transgene CD4+ T-Zellen im Vergleich zu TCR-transduzierten CD8+ T-Zellen eine deutlich verringerte, aber noch erhaltene Funktionalit��t aufwiesen. Es ist derzeit nicht klar, ob HLA-unabh��ngige T-Zellen gegen CSF2RA im peripheren Blut der Patientin vorkamen, weil sie der im Tiermodell postulierten Thymusselektion MHC-unabh��ngiger TCR (Tikhonova et al., Immunity 36:79, 2012) entkommen waren, oder weil ein urspr��nglich gegen einen HLA-Peptid-Komplex gerichteter TCR eine HLA-unabh��ngige Kreuzreaktivit��t aufwies. CSF2RA verbessert die Glucoseutilisation in malignen Zellen, und es wurden ihm embryotrophe Eigenschaften zugeschrieben (Spielholz et al., Blood 85:973, 1995; Sj��blom et al., Biol. Reprod. 67:1817, 2002). Damit kann CSF2RA malignes Wachstum f��rdern und ist somit ein potentielles Zielmolek��l f��r die Immuntherapie. Seine HLA-unabh��ngige Erkennung w��rde sowohl die HLA-Vielfalt als auch den HLA-Verlust als typische Limitationen der T-Zellimmuntherapie umgehen. Zur ��berpr��fung der In vivo-Spezifit��t des HLA-unabh��ngigen TCR gegen CSF2RA und damit zum Ausschluss relevanter off-tumor-/on-target- bzw. off-tumor-/off-target-Effekte ist jedoch eine Testung in einem pr��klinischen Tiermodell erforderlich.

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Evaluation of histological, serological and clinical changes in response to abatacept treatment of primary Sj��gren's syndrome: A pilot study

Objectives To prospectively evaluate histopathologic, blood cellular, serological and clinical changes in response to abatacept treatment in patients with primary Sj��gren's syndrome (pSS). Methods Blood, saliva and minor salivary gland biopsies were obtained before and after the last of 8 doses of abatacept in 11 pSS patients. The histologic data evaluated the number of lymphocytic foci and of B- and T-cell subtypes (CD20(+) , CD3(+) , CD4(+) , CD8(+) ). The numbers of FoxP3(+) regulatory T-cells were measured and the FoxP3 /CD 3 ratio was calculated. Histologic data were compared with results from peripheral blood and with changes in saliva secretion. Results The numbers of lymphocytic foci decreased significantly (p=0.041). Numbers of local FoxP3(+) T-cells decreased significantly in percentage of total lymphocytic infiltrates (p=0.037). In the peripheral blood B-cells increased (p=0.038). This was due to an expansion of the na��ve B cell pool (p=0.034). When adjusting for disease duration, an increase was also noted for total lymphocytes (p=0.044) and for CD 4 cells (p=0.009). Gamma globulins decreased significantly (p=0.005), but IgG reduction did not reach significance. Adjusted for disease duration, saliva production increased significantly (p=0.029). Conclusions CTLA4-Ig treatment significantly reduces glandular inflammation in pSS, induces several celluar changes and increases saliva production. Remarkably, this increase in saliva production is significantly influenced by disease duration.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 ��� T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Thymocyte development is normal in CTLA-4-deficient���mice

Recent studies indicate that CTLA-4 interaction with B7 ligands transduces an inhibitory signal to T lymphocytes. Mice homozygous for a null mutation in CTLA-4 have provided the most dramatic example of the functional importance of CTLA-4 in vivo. These animals develop a fatal lymphoproliferative disorder and were reported to have an increase in CD4+ and CD8+ thymocytes and CD4���CD8��� thymocytes, and a decrease in CD4+CD8+ thymocytes. Based on these observations, it was proposed that CTLA-4 is necessary for normal thymocyte development. In this study, CTLA-4-deficient mice carrying an insertional mutation into exon 3 of the ctla-4 gene were generated. Although these mice display a lymphoproliferative disorder similar to previous reports, there was no alteration in the thymocyte profiles when the parathymic lymph nodes were excluded from the thymi. Further, thymocyte development was normal throughout ontogeny and in neonates, and there was no increase in thymocyte production. Finally, T cell antigen receptor signaling, as assessed by proximal and distal events, was not altered in thymocytes from CTLA-4���/��� animals. Collectively, these results clearly demonstrate that the abnormal T cell expansion in the CTLA-4-deficient mice is not due to altered thymocyte development and suggest that the apparent altered thymic phenotype previously described was due to the inclusion of parathymic lymph nodes and, in visibly ill animals, to the infiltration of the thymus by activated peripheral T cells. Thus it appears that CTLA-4 is primarily involved in the regulation of peripheral T cell activation.

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor���signaling

An early stage in thymocyte development, after rearrangement of the �� chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4���CD8��� double negative (DN) cells to CD4+CD8+ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo �� chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient���mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Retinoid-related orphan receptor �� (ROR��) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor �� (ROR��), a member of the nuclear receptor superfamily, mice deficient in ROR�� function were generated by targeted disruption. ROR�����/��� mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that ROR�� expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of ROR�����/��� mice contains 74.4% �� 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of ROR�����/��� mice. The latter was supported by the observed increase in annexin V-positive cells. ROR�����/��� thymocytes placed in culture exhibit a dramatic increase in the rate of ���spontaneous��� apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from ROR�����/��� mice. Our observations indicate that ROR�� is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which ROR�� negatively controls apoptosis in thymocytes.

Bclx regulates the survival of double-positive thymocytes.

The bclx gene has been shown to regulate programmed cell death in vitro. We now show that Bclx expression increases dramatically when T cells differentiate from CD4- CD8- (double negative) thymocytes to CD4+ CD8+ [double positive (DP)] thymocytes. In contrast single-positive (SP) thymocytes express negligible amounts of Bclx protein. This expression pattern contrasts with that of Bcl2, which is present in double-negative thymocytes, down-regulated in DP thymocytes, and reinduced upon maturation to SP thymocytes. Elimination of Bclx by gene targeting dramatically shortens the survival of DP thymocytes but not the survival of SP thymocytes or peripheral SP T cells. These data suggest that the induction of Bclx during thymic maturation plays a critical role in regulating the length of time DP thymocytes survive in the absence of selection.

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.