Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.

The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.

Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

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.

Endothelial Progenitor Cells in Breast Cancer

Editorial

Concise review:bone marrow for the treatment of spinal cord injury: mechanisms and clinical applications

Transplantation of bone marrow stem cells into spinal cord lesions enhances axonal regeneration and promotes functional recovery in animal studies. There are two types of adult bone marrow stem cell; hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). The mechanisms by which HSCs and MSCs might promote spinal cord repair following transplantation have been extensively investigated. The objective of this review is to discuss these mechanisms; we briefly consider the controversial topic of HSC and MSC transdifferentiation into central nervous system cells but focus on the neurotrophic, tissue sparing, and reparative action of MSC grafts in the context of the spinal cord injury (SCI) milieu. We then discuss some of the specific issues related to the translation of HSC and MSC therapies for patients with SCI and present a comprehensive critique of the current bone marrow cell clinical trials for the treatment of SCI to date.

Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury

Carbon monoxide (CO) has emerged as a vascular homeostatic molecule that prevents balloon angioplasty-induced stenosis via antiproliferative effects on vascular smooth muscle cells. The effects of CO on reendothelialization have not been evaluated.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.

Continuous acquisition of MHC:peptide complexes by recipient cells contributes to the generation of anti-graft CD8+T cell immunity

Understanding the evolution of the direct and indirect pathways of allorecognition following tissue transplantation is essential in the design of tolerance-promoting protocols. On the basis that donor bone marrow-derived antigen presenting cells are eliminated within days of transplantation, it has been argued that the indirect response represents the major threat to long term transplant survival, and is consequently the key target for regulation. However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells, led us to propose a third, semi-direct, pathway of MHC allorecognition. Persistence of this pathway would lead to sustained activation of direct pathway T cells, arguably persisting for the life of the transplant. In this study, we focused on the contribution of acquired MHC class I, on recipient DCs, during the life span of a skin graft. We observed that MHC class I acquisition by recipient DCs occurs for at least one month following transplantation and may be the main source of alloantigen that drives CD8+ cytotoxic T cell responses. In addition, acquired MHC class I-peptide complexes stimulate T cell responses in vivo further emphasizing the need to regulate both pathways to induce indefinite survival of the graft.

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of beta-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost-and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Small clonal B-cell population in the bone marrow as a possible tool in the diagnosis of occult primary parotid lymphoma

Case Description: An 82-years old Hispanic woman with a past medical history significant for pulmonary thromboembolism on oral anticoagulation, rheumatoid arthritis, and hypertension developed a new onset thrombocytopenia. Clinical Findings: Small clonal B-cells populations (SCBP) also known as monoclonal B-cell lymphocytosis was found as part of the workup for an idiopathic thrombocytopenia and lead ultimately to the diagnosis of parotid primary follicular lymphoma coexisting with Warthin tumor involving the bone marrow in a small extent and oncocytic papilloma located in the maxillary sinus. Treatment and Outcome: Patient was treated with Rituximab monotherapy with improvement on her platelet count. Clinical relevance: Although it is unclear the role of this clonal cells, they may work as a possible diagnostic tool for occult lymphomas. Further prospective studies are needed to confirm this possible association.

Roles of the Immune Cytokine Environment on Clonal Growth of Murine and Human Tet2 Mutant Bone Marrow Progenitors: Implications for Myelodysplastic Syndromes

TET2 is a tumor suppressor gene that has been implicated in the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in MDS. These mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Common to the environment of MDS are elevations in cytokines, such as TNFα and IFN-γ. It was hypothesized that inflammatory cytokines TNF-α and IFN-γ may promote clonal expansion of TET2 mutant progenitors. Adult (10-14 weeks-old) Tet2 wild type (+/+) and Tet2 mutant (-/-) C57BL/6 mice strains were chosen as a model system. Lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells, were isolated from Tet2 +/+ and -/- bone marrow and cultured in the absence or presence of varying concentrations of TNFα or IFN-γ in methylcellulose colony formation assays and long term cell culture assays, over a period of 12 and 30 days respectively, and their colony growth, cell count, immunophenotype and resistance to apoptosis were examined. Where indicated, serial re-plating was performed. Expression of apoptotic regulators was assessed by qRT-PCR. In the triplicate experiments, starting with equal densities of Tet2 +/+ and -/- Lin- cells, Tet2 -/- Lin- cells displayed increased resistance to cytokine-induced growth suppression and superior colony forming ability over +/+ in the serial re-plating assays under stress of increasing TNFα or IFN γ. Tet2 -/- progenitors also displayed a lower apoptotic index compared to +/+ under stress of increasing TNFα, suggesting increased resistance to TNFα induced apoptosis. Transcriptional data showed low expression of Tnfr1, Fas and caspase 8, as well as a high expression of Bcl-2 and Iap1 in Tet2 -/- compared to +/+ under stress of TNFα. Tet2-/- also showed increased basal expression of endogenous TNFα mRNA compared to +/+. In the human colony growth assay, the clonal growth of TET2 mutant CFU-GM progenitors was enhanced at low TNFα concentrations. Conclusion: Mutations that promote resistance to environmental stem cell stressors are a known mechanism of clonal selection in aplastic anaemia and JAK2-mutant MPN and our findings suggest that this mechanism may be critical to clonal selection and dominance in MDS.