Analysis of potential transcriptomic biomarkers for Huntington's disease in peripheral blood.

Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntington's disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.

An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations.

Is peripheral blood cell balanced altered by the use of fresh frozen bone block allografts in lateral maxillary ridge augmentation?

Background: The relationship between the immune response and red and white blood cell homeostasis is cited in literature, but no studies regarding the balance of these cell populations following maxillary bone-graft surgeries can be found. Aim: The aim of this study was to evaluate the possible impairments in the blood cell balance following fresh-frozen allogeneic bone-graft augmentation procedures in patients who needed maxillary reconstruction prior to implants. Material and Methods: From 33 patients elected to onlay bone grafting procedures, 20 were treated with fresh-frozen bone allografts and 13 with autologous bone grafts. Five blood samples were collected from each patient in a 6-month period (baseline: 14, 30, 90, and 180 days postsurgery), and the hematological parameters (erythrogram, leukogram, and platelets count) were accessed. Results: All evaluated parameters were within the reference values accepted as normal, and significant differences were found for the eosinophils count when comparing the treatments (30 days, p=.035) and when comparing different periods of evaluation (allograft-treated group, baseline×180 days, p≤.05 and 90×180 days, p≤.01; autograft-treated group, 30×90 days, p≤.05 and 30×180 days, p≤.05). Conclusions: Both autologous and fresh-frozen allogeneic bone grafts did not cause any impairment in the red and white blood cell balance, based on quantitative hemogram analysis, in patients subjected to maxillary reconstruction. © 2011 Wiley Periodicals, Inc.

Patients With Endometriosis of the Rectosigmoid Have a Higher Percentage of Natural Killer Cells in Peripheral Blood

Study Objective: To estimate the concentration of natural killer (NK) cells in the peripheral blood in patients with and without endometriosis. Design: Case-control study (Canadian Task Force classification II-2). Setting: Tertiary referral hospital. Patients: One hundred fifty-five patients who had undergone videolaparoscopy were divided into 2 groups: those with endometriosis (n = 100) and those without endometriosis (n = 55). Interventions: The percentage of NK cells relative to peripheral lymphocytes was quantified at flow cytometry in 155 patients who had undergone laparoscopy. In addition to verifying the presence of endometriosis, stage of disease and the sites affected were also evaluated. Measurements and Main Results: The mean (SD) percentage of NK cells was higher (15.3% [9.8%]) in patients with endometriosis than in the group without the disease (10.6% [5.8%]) (p < .001). The percentage of NK cells was highest (19.8 [10.3%]) in patients with advanced stages of endometriosis and in those in whom the rectosigmoid colon was affected. In a statistical model of probability, the association of this marker (NK cells >= 11%) with the presence of symptoms such as pain and intestinal bleeding during menstruation and the absence of previous pregnancy yielded a 78% likelihood of the rectosigmoid colon being affected. Conclusion: Compared with patients without endometriosis, those with endometriosis demonstrate a higher concentration of peripheral NK cells. The percentage of NK cells is greater, primarily in patients with advanced stages of endometriosis involving the rectosigmoid colon. Therefore, it may serve as a diagnostic marker for this type of severe endometriosis, in particular if considered in conjunction with the symptoms. Journal of Minimally Invasive Gynecology (2012) 19, 317-324 (C) 2012 AAGL. All rights reserved.

Identification of preferentially targeted tumor-associated antigens in melanoma patients via mRNA stimulation of CD8+ blood lymphocytes

Until now, therapeutic vaccination of cancer patients has mainly relied on rather few T cell epitopes processed from structurally normal shared tumor antigens and presented by frequent HLA alleles. So far the design of these studies has not addressed the individuality of tumor-host interactions, which are not only determined by the antigenic tumor phenotype or the natural HLA polymorphism, but also by the individual T cell repertoire. The procedure described herein was developed to identify the preferential targets of the individual repertoire from a panel of known shared tumor-associated antigens. Lymphocytes were isolated from the peripheral blood of cancer patients or healthy donors and stimulated twice with autologous mRNA-transfected FastDC (Dauer et al., J Immunol. 170:4069, 2003). FastDC were generated from blood monocytes and separately transfected via lipofection with in vitro transcribed mRNAs encoding the panel antigens. Responder lymphocytes were tested on day 12 in a 20-hour IFN-g ELISPOT assay for recognition of 293T cells co-transfected pairwise with plasmids encoding the stimulation antigens and the respective individual’s HLA class I alleles. In a first step, stimulation parameters were optimized for the detection of anti-HCMV pp65 responses. A maximum amplification of pp65-specific CD8+ T cell responses was obtained at a rather low IL-2 concentration (25 IU/ml) and at a minimum APC-to-effector ratio of 1:10. Addition of IL-4, IL-7 or IL-15 did not substantially improve the stimulatory potential. The test was applied to the human melanoma models D05 and MZ2, in both of which multiple T cell-defined antigens had previously been identified by expression screening. Blood lymphocytes were stimulated in parallel with autologous tumor cells and with mRNA-transfected FastDC. In D05, T cell reactivities against three out of eleven epitopes induced by stimulation with tumor cells were also found after stimulation with mRNA-transfected FastDC. Two further T cell target epitopes were identified with mRNA but not with tumor cell stimulation. In MZ2, T cell responses against five distinct epitopes were detected on day 12 after stimulation with mRNA transfectants. The same responses were detectable after stimulation with tumor cells only on day 32. mRNA stimulations against 21 tumor-associated antigens in addition to HCMV pp65 were performed in four healthy individuals. In all cases, CD8+ T cells against HCMV pp65 could be expanded. Among tumor-associated antigens, only reactivity against Melan-A/MART-1 in association with HLA-A*0201 was detectable in one of the donors. The vaccination of patients with targets a priori known to be recognized by their T cell repertoire may help to improve the outcome of therapeutic vaccination.

The roles of costimulatory molecules in the cooperative effects of combination epinephrine and dexamethasone on type-1/type-2 cytokine production in tetanus-toxoid specific stimulated human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high circulating levels of catecholamines (CT) and corticosteroids (CS) that are associated with changes in type-1/type-2 cytokine expression. Although individual pharmacologic doses of CS and CT can inhibit the expression of T-helper 1 (Th1, type-1 like) and promote the production of T-helper 2 (Th2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination physiologic-stress doses of CT and CS that may be more physiologically relevant. In addition, both in-vivo and in-vitro studies suggest that the differential expression of the B7 family of costimulatory molecules CD80 and CD86 may promote the expression of type-1 or type-2 cytokines, respectively. Furthermore, corticosteroids can influence the expression of β2-adrenergic receptors in various human tissues. We therefore investigated the combined effects of physiologic-stress doses of in vitro CT and CS upon the type-1/type-2 cytokine balance and expression of B7 costimulatory molecules of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of physiologic stress. Results demonstrated a significant decrease in type-1 cytokine expression and a significant increase in type-2 cytokine production in our CS+CT incubated cultures when compared to either CT or CS agents alone. In addition, we demonstrated the differential expression of CD80/CD86 in favor of CD86 at the cellular and population level as determined by flow cytometry in lipopolysaccharide stimulated human Monocytes. Furthermore, we developed flow cytometry based assays to detect total β2AR in human CD4+ T-lymphocytes that demonstrated decreased expression of β2AR in mitogen stimulated CD4+ T-lymphocytes in the presence of physiologic stress levels of CS and CT as single in vitro agents, however, when both CS and CT were combined, significantly higher expression of β2AR was observed. In summary, our in vitro data suggest that both CS and CT work cooperatively to shift immunity towards type-2 responses. ^

Direct visualization of antigen-specific T cells: HTLV-1 Tax11–19- specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11–19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2–Ig complex to directly visualize HTLV-1 Tax11–19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+ lymphocytes specific for the HTLV-1 Tax11–19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11–19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11–19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11–19-specific CD8+ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-α and γ-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11–19-specific CD8+ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.

We have investigated the ability of human immunodeficiency virus (HIV)-infected cells to kill uninfected CD4+ lymphocytes. Infected peripheral blood mononuclear cells were cocultured with autologous 51Cr-labeled uninfected cells. Rapid death of the normal CD4-expressing target population was observed following a brief incubation. Death of blood CD4+ lymphocytes occurred before syncytium formation could be detected or productive viral infection established in the normal target cells. Cytolysis could not be induced by free virus, was dependent on gp120-CD4 binding, and occurred in resting, as well as activated, lymphocytes. CD8+ cells were not involved in this phenomenon, since HIV-infected CEMT4 cells (CD4+, CD8- cells) mediated the cytolysis of uninfected targets. Reciprocal isotope-labeling experiments demonstrated that infected CEMT4 cells did not die in parallel with their targets. The uninfected target cells manifested DNA fragmentation, followed by the release of the 51Cr label. Thus, in HIV patients, infected lymphocytes may cause the depletion of the much larger population of uninfected CD4+ cells without actually infecting them, by triggering an apoptotic death.

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

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.

Significant alterations in peripheral blood lymphocyte subsets in patients with somatoform disorder

Objective: Previous studies have suggested that somatoform disorders (SFD) might be associated with changes in the function of the central and autonomic nervous systems. The aim of this study was to examine the possible immunological differences between SFD and healthy controls. Methods: Twenty-four patients with SFD and 13 healthy individuals completed the psychological questionnaires to assess symptom reporting [Symptom Checklist-90 Revised (SCL-90-R)] and to diagnose for SFD [Screening for Somatoform Symptoms scale (SOMS-scale)]. Participants also provided a blood sample taken in the morning, which was analysed with an automated cell counter to determine the number of leucocytes per μl and with flow cytometry to determine lymphocyte subsets. Results: With the exception of a higher T4/T8 ratio in the patient group, which was mainly because of lower CD8 counts, there were no significant differences in the absolute number of lymphocytes (subsets) between patients with SFD and healthy subjects. A positive correlation between B-lymphocyte subsets (CD19+CD22+, CD19+CD5+, CD19+CD3-) to all scales of the SCL-90-R, except somatisation, were found in SFD. Additionally, a positive correlation was found in SFD between CD14+CD16+ monocytes and somatisation (0.573) on the SCL-90-R scale. Conclusion: These data indicate that patients with SFD have an enhanced humoral immunity as shown by increased B-cell numbers and furthermore an elevated T4/T8 ratio because of lower CD8 suppressor cells. Further studies will be required to determine whether these alterations in lymphocyte subsets are directly involved in the pathophysiology of SFD. © 2007 Blackwell Munksgaard.

ABCA1 expression and statins: inhibitory effect in peripheral blood mononuclear cells

The ATP-binding cassette transporter A1 (ABCA1) has an essential role in the formation of nascent high-density lipoprotein particles and also participates in the cholesterol efflux from macrophages in the artery wall. Several substances, such as statins, or even gene variants are able to modulate ABCA1 expression. There is strong evidence that statin treatment downregulates the ABCA1 expression in nonloaded macrophages. Interestingly, in cholesterol-loaded macrophages, which are more relevant to atherogenesis, this effect is lost. We observed an inhibitory effect of atorvastatin in peripheral blood mononuclear cells of hypercholesterolemic individuals. Moreover, in these individuals, the ABCA1 -14C > T polymorphism was associated with high baseline gene-expression levels. Other studies are needed to evaluate how relevant these findings are to the formation of arterial foam cells in vivo.

Peripheral blood chimerism following human liver transplantation

The aim of this study was to prospectively investigate the peak levels and kinetics of donor leucocyte chimerism in human recipients following liver transplantation, The peak levels of chimerism mere observed within the first 48 hours following transplantation and ranged from 0.15% to 20% of total peripheral blood mononuclear cells, In all but one patient, who developed graft versus host disease, there was an early peak level of chimerism that declined over time such that donor leukocytes mere only intermittently detectable after 3 to 4 weeks. In 8 patients who had no episodes of graft rejection, the peak level of donor leukocyte chimerism ranged from 1.3% to 20% (mean +/- SEM; 5.5% +/- 2.1%). In 3 patients who were treated for episodes of acute graft rejection during the first four postoperative weeks, the peak level of donor leukocyte chimerism ranged from 0.15% to 0.2% (0.18 +/- 0.02, P = .012), The results demonstrate a marked variation in the total number of donor leukocytes detectable in the peripheral blood early after liver transplantation and also, that lower levels of chimerism may be associated with lower rates of initial graft acceptance and a higher incidence of acute rejection.