Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Autologous implant of bone marrow mononuclear cells as treatment of induced equine tendinitis

Superficial digital flexor tendonitis is an important cause of lameness in horses and its incidence ranges from 13% to 30%, depending on the horse's activity. This injury can occur in yearlings and compromise its carriers by reinjury or even impossibility to return to athletic life. In spite of the long period required for tendon repair, the scar tissue presents lack of elasticity and stiffness. As current treatment strategies produce only marginal results, there has been great interest in research of therapies that influence the quality or the speed of tendon repair. Stem cell therapy has shown promising results in degenerative diseases and cases of deficient healing processes. This study aims to evaluate the influence of autologous mesenchymal bone marrow stem cells in tendon healing, comparing treated and non-treated tendons. Superficial digital flexor tendonitis lesions were induced by collagenase infiltration in both forelimbs of 6 horses, followed by autologous implant in one of the forelimbs of each animal. The horses were evaluated using clinical, ultrasonographic, histopathologic, and immunohistochemical parameters. Tendon biopsies were performed at Day 48. Results found in the treatment group, such as high inflammatory cells infiltration, extracellular matrix synthesis, reduced amount of necrosis areas, small increase in cellular proliferation (KI-67/MIB-1), and low immunoreactivity to transforming growth factor P I, suggested the acceleration of tendon repair in this group. Further studies should be conducted in order to verify the influence of this treatment on later phases of tendon repair. Overall, after analysis of the results, we can conclude that cellular therapy with the mononuclear fraction of bone marrow has accelerated tendon repair at 48 days after treatment.

Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento

Cinco cavalos adultos foram submetidos à coleta de medula óssea do esterno e de tecido adiposo da região glútea. As amostras foram processadas para obtenção da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo, o número de células obtidas e a viabilidade celular foram determinados. Em seguida, realizou-se o congelamento das amostras em solução contendo 20% de soro fetal bovino e 10% de dimetilsulfóxido. Depois de um mês, realizou-se o descongelamento das amostras e a viabilidade celular foi novamente mensurada. Os resultados revelaram que as técnicas utilizadas tanto para coleta de medula óssea quanto de tecido adiposo em equinos são simples, rápidas e seguras. As metodologias adotadas para o processamento das amostras foram eficientes, obtendo-se aproximadamente 95% de viabilidade celular. Após o descongelamento, a viabilidade média das amostras de células mononucleares da medula óssea foi de 86% e da fração vascular estromal do tecido adiposo de 64%. Frente à importância da terapia celular na clínica médica de equinos, concluiu-se que é necessária a realização de mais estudos, visando padronizar uma técnica de criopreservação que mantenha a integridade das células da fração mononuclear da medula óssea e da fração vascular estromal do tecido adiposo de equinos.

Quantificação das células estreladas ativadas / miofibroblastos e análise da apoptose das células do fígado durante a terapia celular na fibrose hepática em ratos

A fibrose hepática é o resultado de uma resposta cicatrizante frente a repetidas lesões no fígado, e é caracterizada pelo acúmulo excessivo de proteínas da matriz extracelular (MEC) no parênquima hepático, incluindo colágeno, fibronectina, elastina, laminina e proteoglicanos, com a participação de diferentes populações celulares do fígado. As principais células responsáveis pela síntese de proteínas da MEC na fibrose hepática são as células estreladas hepáticas ativadas e os miofibroblastos, que surgem após estímulo inflamatório e são caracterizadas pela expressão de alfa-actina de músculo liso (α-SMA). Sabe-se que durante a progressão da fibrose hepática, ocorre a morte de hepatócitos e sua substituição por células fibrogênicas α-SMA+. A apoptose dessas células fibrogênicas é de grande relevância para a regressão da fibrose e regeneração hepática. Nos últimos anos, a terapia com células tronco de medula óssea tem sido utilizada para estimular a regeneração hepática em diferentes modelos experimentais e protocolos clínicos. A fração mononuclear da medula óssea adulta possui duas populações de células-tronco importantes no tratamento de diversas doenças hepáticas: células-tronco hematopoiéticas e células-tronco mesenquimais. O objetivo deste estudo foi analisar a expressão de α-SMA e o processo de apoptose de células hepáticas durante a fibrose hepática induzida por ligadura do ducto biliar (LDB) e após o transplante de células mononucleares de medula óssea (CMMO). Os fígados foram coletados de ratos dos seguintes grupos: normal, 14 dias de LDB, 21 dias de LDB e animais que receberam CMMO após 14 dias de LDB, e foram analisados após 7 dias (totalizando 21 dias de LDB). Para quantificar a expressão de α-SMA por células fibrogênicas nos grupos experimentais, foi realizada imunoperoxidase para α-SMA, seguida de morfometria no programa Image Pro Plus. Para analisar a apoptose nas células hepáticas, foi realizada imunoperoxidase e Western Blotting (WB) para caspase-3 (proteína apoptótica) e imunofluorescência com dupla-marcação para caspase-3 e α-SMA, seguida de observação em microscópio confocal. Os resultados da quantificação de α-SMA por morfometria mostraram que a expressão de α-SMA aumentou significativamente 14 e 21 dias após a LDB. Entretanto, essa expressão diminuiu significativamente no grupo tratado com CMMO, que apresentou parênquima hepático mais preservado em relação ao grupo com 21 dias de LDB. Os resultados de imunoperoxidase, WB e microscopia confocal para expressão de caspase-3 demonstraram que essa proteína diminuiu nos animais fibróticos com 14 e 21 dias de LDB com relação ao grupo normal, e estava significativamente elevada no grupo tratado com CMMO. A análise por microscopia confocal demonstrou que algumas células coexpressaram α-SMA e caspase-3 nos animais tratados com CMMO, sugerindo a morte de células fibrogênicas e remodelamento do parênquima hepático.

Uso de células mononucleares da medula óssea no tratamento de tendinites induzidas experimentalmente em equinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Células mononucleares da medula óssea no tratamento da ruptura total dos tendões flexores e ligamento suspensor do boleto em equino

This paper describes the case of a horse treated in a Teaching Veterinary Hospital. The sutures of the flexor apparatus associated with cell therapy, the stem cell, provided a rapid response of repair compared to other cases treated in routine of the Hospital. The association of conventional treatments with the mononuclear fraction of bone marrow significantly contributed to the reduction of the hyperextension of the metatarsophalangeal joint. Ultrasound images showed rapid fill cell and decrease in inflammation.

Implante autólogo de células mononucleares provenientes de medula óssea notratamento de tendinites induzidas experimentalmente em equinos: avaliação a longo prazo - 120 dias

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone Marrow'S Harvest in The Coxal Tuberosity for Isolation and Culture of Mesenchymal Stem Cells of Buffaloes (Bubalus Bubalis)

Several studies with mesenchymal stem cells (MSCs) have been developed in many species because of its ability to differentiate into other mesoderm lineages, capacity of self-regeneration, low immunogenicity, paracrine, anti-inflamatory, immunomodulatory and antiapoptotic effects which make then a promissory source to be used in therapeutic strategies. The aim of this study is to report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes. For this, the animals were selected, identified and contained in a stock. Then trichotomy was performed in the region corresponding to the CT. After identifying the anatomic site it was performed antisepsis, local anesthetic block and introduction of a myelogram's needle (Lang(R)) for BM aspiration. Once the needle was firmly fixed in the CT, the mandril was removed and proceeded to BM aspiration with a syringe (20 mL) containing 1 ml of heparin at 1000 IU / mL and 1 mL of PBS. After the collection, each sample collected was manually homogenized, identified and referred to the LRACT - FMVZ / UNESP-BRAZIL for the correct processing. The anatomical site tested showed to be an alternative site of harvest of BM once provided the appropriate isolation and culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and with great security. Based on this, it can be conclude that CT is an excellent anatomical site for isolation and culture of MSCs and the proposed technique is viable and feasible to be held in buffaloes.

Uso de células-tronco para o tratamento de tendinite em eqüinos

Brazil has the fourth largest horse herd in the world, this is due the recognition and appreciation that the different equestrian games are having within the country. Injuries of the tendon, especially in the digital flexor tendon, are the main cause of athletic life reduction among horses. The treatment of tendinitis in horses seeks full recovery of the damage tissue reestablishing the function previously lost, however conventional treatments have proven to be ineffective when considered the quality of the scar tissue and the rate of recurrence. Due to this, the use of adult stem cells to the treatment of musculoskeletal injuries of horses has been studied for some time. This method of treatment consists of aspiration of bone marrow or removal of subcutaneous fat tissue and implantation of these cells in the injured tissue. After obtaining the bone marrow the implantation can be performed with total bone marrow, with the mononuclear fraction of MSC or with cells cultured in vitro. From the fat tissue is used the stromal vascular fraction obtained by collagenase digestion, followed or not by cell culture. According to some studies, cell therapy with material obtained from bone marrow or adipose tissue has shown to be viable, given that these materials are abundant in repair components such as mesenchymal stem cells (MSC), growth factors and other components of the collagen matrix. Several studies using both types of cells have shown great potential and promising clinical results. However, knowledge of the biology and characterization of these cells remain largely unknown, and therefore is needed great care and caution when using stem cells for the treatment of musculoskeletal disorders in horses

Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells

Olive pomace oil, also known as "orujo" olive oil, is a blend of refined-pomace oil and virgin olive oil, fit for human consumption. Maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes, found in the non-glyceride fraction of orujo oil, which have previously been reported to have anti-inflammatory properties. In the present work, we investigated the effect of these minor components on pro-inflammatory cytokine production by human peripheral blood mononuclear cells in six different samples. Uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1 beta and IL-6 production in a dose-dependent manner. All three compounds significantly reduced TNF-alpha production at 100 mu M; however, at 10 mu M, uvaol and oleanolic acid enhanced the generation of TNF-alpha. In contrast, maslinic acid did not significantly alter the concentration of those cytokines, with the exception of a slight inhibitory effect at 100 mu M. All four triterpenes inhibited production of I-309, at 50 mu M and 100 mu M. However, uvaol enhanced I-309 production at 10 mu M. The triterpenic dialcohols had a similar effect on MIG production. In conclusion, this study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and anti-inflammatory properties depending on chemical structure and dose, and may be useful in modulating the immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Cardiomyopathy and cell therapy: ejection fraction improvement and cardiac muscle mass increasing, after a year of bone marrow stem cells transplantation, by magnetic resonance image

The idiopathic dilated cardiomyopathy (IDC) is one of the major public health problems in the western world. Patients with IDC in functional class IV (New York Health Association - NYHA), even after therapeutic optimization, have high mortality. Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases and several positive effects were assigned to cell therapy in cardiomyopathy. The aim of this study was identify short-term result of cell transplantation in idiopathic dilated cardiomyopathy patients (IDC) who were treated by transplantation of autologous bone marrow mononuclear cells (BMMC). Intracoronary injections of autologous BMMC were performed in eight patients with severe ventricle dysfunction (mean of left ventricle ejection fraction – LEVF=20.03%), cardiac mass muscle around 156.2 g and NYHA between III and IV grades, other 8 IDC patients received placebo. The IDCs were followed - up for one and two years, by magnetic resonance imaging (MRI). The results after one year showed significant improvement in LVEF (mean=181.4) and muscle mass increasing (mean=181.4 g), after two years the LVEF continued improving, reaching a mean of 32.69% and the cardiac muscle mass kept stable (mean=179.4 g). Excepted for one patient, all the other had improvement in the NYHA functional class. The placebo group did not show any improvement. We believe that BMMC implant may be a beneficial therapeutic option for IDC patients.

Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function

BACKGROUND Intracoronary administration of autologous bone marrow-derived mononuclear cells (BM-MNC) may improve remodeling of the left ventricle (LV) after acute myocardial infarction. The optimal time point of administration of BM-MNC is still uncertain and has rarely been addressed prospectively in randomized clinical trials. METHODS AND RESULTS In a multicenter study, we randomized 200 patients with large, successfully reperfused ST-segment elevation myocardial infarction in a 1:1:1 pattern into an open-labeled control and 2 BM-MNC treatment groups. In the BM-MNC groups, cells were administered either early (i.e., 5 to 7 days) or late (i.e., 3 to 4 weeks) after acute myocardial infarction. Cardiac magnetic resonance imaging was performed at baseline and after 4 months. The primary end point was the change from baseline to 4 months in global LV ejection fraction between the 2 treatment groups and the control group. The absolute change in LV ejection fraction from baseline to 4 months was -0.4±8.8% (mean±SD; P=0.74 versus baseline) in the control group, 1.8±8.4% (P=0.12 versus baseline) in the early group, and 0.8±7.6% (P=0.45 versus baseline) in the late group. The treatment effect of BM-MNC as estimated by ANCOVA was 1.25 (95% confidence interval, -1.83 to 4.32; P=0.42) for the early therapy group and 0.55 (95% confidence interval, -2.61 to 3.71; P=0.73) for the late therapy group. CONCLUSIONS Among patients with ST-segment elevation myocardial infarction and LV dysfunction after successful reperfusion, intracoronary infusion of BM-MNC at either 5 to 7 days or 3 to 4 weeks after acute myocardial infarction did not improve LV function at 4-month follow-up.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

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.