Apheresis and Cell Therapy Unit

Pavia, Italy

Apheresis and Cell Therapy Unit

Pavia, Italy
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Caramella C.M.,University of Pavia | Sandri G.,University of Pavia | Rossi S.,University of Pavia | Mori M.,University of Pavia | And 4 more authors.
Current Drug Delivery | Year: 2013

There are still so-called unmet needs in the treatment of epithelial and cutaneous lesions. Mucositis, ocular lesions, chronic skin wounds represent typical examples. These pathologies do not yet afford a satisfactory treatment. In particular chronic wounds represent a major health care burden, likely to increase as the population ages. Healing of epithelial and cutaneous lesions progresses through a complex cascade of events starting with the secretion in the local environment of a pool of growth factors, cytokines and proteins from the serum and degranulating platelets. Recently platelet lysate (PL), a hemoderivative obtained by platelet destruction by freeze-thawing of a platelet rich plasma (PRP) sample in the presence of an anticoagulant agent, has proved capable of promoting the healing of buccal and corneal lesions. Since the efficacy of growth factors (GFs) critically depends on the way they are made available to the injured tissue, the development of suitable therapeutic vehicles is of paramount importance to release GFs according to the repairing requirements. The present work focuses on the development and testing of few such formulations, in particular a mucoadhesive gel and an in situ gelling buccal spray for the treatment of oral mucositis, and a thermosensitive eye drop solution for corneal lesions. Besides technological characterization, the formulations have been preliminarily screened with an ELISA assay, a cell proliferation test and an in vitro wound healing test. Some formulations have been used in early clinical trials. © 2013 Bentham Science Publishers.


Sandri G.,University of Pavia | Bonferoni M.C.,University of Pavia | Ferrari F.,University of Pavia | Rossi S.,University of Pavia | And 4 more authors.
Current Drug Discovery Technologies | Year: 2011

Oral mucositis is an inflammatory disease of the mucosa lining oral cavity which leads to atrophy of the epithelium and to its ulceration to form chronic lesions. Many studies, both in vitro and in vivo, have disclosed the effectiveness of growth factors derived from platelets to enhance cell proliferation, differentiation, chemotaxis, angiogenesis and extracellular matrix synthesis involved in the healing of tissues. Despite potential usefulness of growth factors, animalderived or genetically engineered ones are currently scarcely available for regenerative therapies. The aim of the present work was to study an in situ gelling formulation to be delivered by a spraying device to the oral cavity affected by mucositis. A vehicle based on Poloxamer 407 (F127) and sodium alginate (LVG) was developed. An extemporaneous loading of the vehicle with PL was achieved. The formulation was able to quickly thermogelify at 34-35°C with a viscosity at 8°C suitable for spraying; moreover it was characterized by good mucoadhesive properties. ELISA assay evidenced that at time zero the growth factor PDGF AB was compatible with the vehicle. The in vitro wound healing test showed that the formulation enhanced cell growth as PL alone even after 72 h of contact without cell apoptosis. The overall results indicate that PL loaded in the in situ gelling F127 and LVG vehicle can be profitably employed to treat buccal mucositis. © 2011 Bentham Science Publishers.


Sandri G.,University of Pavia | Bonferoni M.C.,University of Pavia | Rossi S.,University of Pavia | Ferrari F.,University of Pavia | And 4 more authors.
International Journal of Pharmaceutics | Year: 2012

Corneal lesions cause significant pain and visual impairment and, in many cases, are unresponsive to conventional treatments. Platelet lysate (PL) is an haemoderivative rich in growth factors (GFs) that are released by platelets after freeze-thawing destruction of platelet rich plasma (PRP). The aim of the present work was to develop thermosensitive and mucoadhesive eyedrops to maintain and prolong the contact of platelet lysate (PL) with cornea ulcers. A sterile vehicle based on chondroitin sulphate sodium (CS) and hydroxypropylmethyl cellulose (HPMC) was developed. An extemporaneous loading of the vehicle with PL was performed and the obtained formulation was able to quickly thermogelify at about 32 °C and was characterized by good mucoadhesive properties. ELISA evidenced that the growth factor PDGF AB was compatible with the vehicle and stable in the formulation up to 15 days of storage at 2-8 °C. In vitro wound healing and proliferation test (performed using rabbit corneal epithelial cells (RCE)) showed that the formulation enhanced cell growth and put in evidence a synergistic effect of CS and PL in stimulating cell proliferation. The overall results indicate that PL loaded in thermosensitive and mucoadhesive eyedrops can be profitably employed to treat corneal lesions. © 2012 Elsevier B.V. All rights reserved.


Perotti C.,Apheresis and Cell Therapy Unit | Del Fante C.,Apheresis and Cell Therapy Unit | Tinelli C.,Apheresis and Cell Therapy Unit | Viarengo G.,Apheresis and Cell Therapy Unit | And 4 more authors.
Transfusion | Year: 2010

BACKGROUND: Extracorporeal photochemotherapy (ECP) is a valid therapeutic option in the treatment of acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively). No standard clinical and laboratory criteria of response to ECP treatment are available at the moment. STUDY DESIGN AND METHODS: Clinical and laboratory variables on 73 pediatric patients with aGVHD (n = 50) and cGVHD (n = 23) were correlated with response to ECP and survival. RESULTS: An overall response (OR) was obtained in 34 of 50 (68%) aGVHD and in 16 of 23 (69.5%) cGVHD patients. Steroid tapering within 30 days of 1.3 mg/kg in OR (p = 0.004) was the sole highly significant correlation with response found in aGVHD while no correlation emerged for cGVHD (p = 0.28). Among aGVHD patients, response to ECP was inversely associated with death: among OR, deaths were 13 of 34 (38.2%), while among nonresponders, deaths were 15 of 16 (93.8%; p < 0.001). On the other hand, decrease of steroid dose at 30 days was associated with survival: for each 1 mg/kg reduction, the hazard ratio was 2.2, and the 95% confidence interval was 1.5 to 3.2 (p < 0.001). No other clinical or laboratory variables statistically associated with survival were found. CONCLUSIONS: Our results demonstrate that steroid tapering within the first 30 days of ECP treatment in aGVHD and response to ECP in acute and chronic GVHD are the only variables influencing response and survival, respectively. © 2010 American Association of Blood Banks.


Sandri G.,University of Pavia | Bonferoni M.C.,University of Pavia | Rossi S.,University of Pavia | Ferrari F.,University of Pavia | And 8 more authors.
Expert Opinion on Drug Delivery | Year: 2015

Introduction: The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems.Methods: The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion.Results: Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure.Conclusion: Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing. © 2014 Informa UK, Ltd.


Del Fante C.,Apheresis and Cell Therapy Unit | Scudeller L.,Apheresis and Cell Therapy Unit | Viarengo G.,Apheresis and Cell Therapy Unit | Bernasconi P.,Apheresis and Cell Therapy Unit | Perotti C.,Apheresis and Cell Therapy Unit
Transfusion | Year: 2012

BACKGROUND: Chronic graft-versus-host disease (cGVHD) classification has recently been improved by the National Institutes of Health (NIH); patients' stratification with those new criteria has implications for patients' prognosis. STUDY DESIGN AND METHODS: To assess whether the NIH consensus classification (NCC) better predicts survival and response to extracorporeal photochemotherapy (ECP), and to identify variables associated with response and survival, we retrospectively analyzed 102 patients with cGVHD reclassified according to NCC treated with ECP (1997-2010) at our center. Cox regression was used in univariate and multivariate models. RESULTS: Of the 102 patients, 64 (62.7%) had classic cGVHD, 24 (23.5%) had overlap cGVHD, and seven (6.9%) patients each had late and persistent acute GVHD. The cumulative ECP-specific follow-up was 2333.3 person-years. Response was complete in 16 (15.7%), partial in 38 (37.3%), minimal in 28 (27.5%), and absent in 20 (19.6%). Of the 22 deaths, 15 (68.2%) occurred among patients with minimal or no response (p = 0.031). The only variables associated with response were nonmyeloablative transplant (hazard ratio, 3.5; 95% confidence interval [CI], 1.36-9.08; p = 0.009), donor lymphocyte infusion (hazard ratio, 2.58; 95% CI, 1.2-5.56; p = 0.015), and lung involvement (hazard ratio, 0.34; 95% CI, 0.12-0.94; p = 0.038). CONCLUSION: ECP is a safe and effective treatment for cGVHD and response to ECP is the only variable that influences survival. We found no correlation between response and NCC clinical subtype, number, or degree of organ involvement, except for lung, or the variables mentioned above. Prospective studies are needed to identify subsets of patients with higher probability of response. © 2012 American Association of Blood Banks.


Sandri G.,University of Pavia | Bonferoni M.C.,University of Pavia | D'Autilia F.,University of Pavia | Rossi S.,University of Pavia | And 7 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2013

The management of difficult to heal wounds can considerably reduce the time required for tissue repairing and promote the healing process, minimizing the risk of infection. Silver compounds, especially silver sulfadiazine (AgSD), are often used to prevent or to treat wound colonization, also in presence of antibiotic-resistant bacteria. However, AgSD has been shown to be cytotoxic in vitro toward fibroblasts and keratinocytes and consequently to retard wound healing in vivo. Recently, platelet lysate (PL) has been proposed in clinical practice for the healing of persistent lesions. The aim of the present work was the development of wound dressings based on AgSD loaded in solid lipid nanoparticles (SLNs), to be used in association with PL for the treatment for skin lesions. SLN were based on chondroitin sulfate and sodium hyaluronate, bioactive polymers characterized by well-known tissue repairing properties. The encapsulation of AgSD in SLN aimed at preventing the cytotoxic effect of the drug on normal human dermal fibroblasts (NHDFs) and at enabling the association of the drug with PL. SLN were loaded in wound dressings based on hydroxypropylmethyl cellulose (HPMC) or chitosan glutamate (CS glu). These polymers were chosen to obtain a sponge matrix with suitable elasticity and softness and, moreover, with good bioadhesive behavior on skin lesions. Dressings based on chitosan glutamate showed antimicrobial activity with and without PL. Even though further in vivo evaluation could be envisaged, chitosan based dressings demonstrated to be a suitable prototype for the treatment for skin lesions. © 2012 Elsevier B.V. All rights reserved.


Malara A.,University of Pavia | Malara A.,Biotechnology Research Laboratories | Currao M.,University of Pavia | Currao M.,Biotechnology Research Laboratories | And 9 more authors.
Stem Cells | Year: 2014

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration. © AlphaMed Press 2013.


Del Fante C.,Apheresis and Cell Therapy Unit | Scudeller L.,Apheresis and Cell Therapy Unit | Viarengo G.,Apheresis and Cell Therapy Unit | Cervio M.,Apheresis and Cell Therapy Unit | Perotti C.,Apheresis and Cell Therapy Unit
Transfusion | Year: 2013

Background Extracorporeal photochemotherapy (ECP) is an effective cell therapy employed in several diseases, including graft-versus-host disease (GVHD) and organ rejection after transplantation. When ECP is performed using the off-line technique, mononuclear cell (MNC) collection by leukapheresis is necessary for further manipulation. Semiautomated apheresis systems require experienced personnel to obtain a good MNC collection; an automated device, able to efficiently collect MNCs with high purity, is desirable. We compared the semiautomated COBE Spectra MNC and the new automated Spectra Optia v.5.0 MNC (Terumo BCT) devices in terms of efficacy and safety. Study Design and Methods Adult patients with GVHD or bronchiolitis obliterans syndrome (BOS) after lung transplant undergoing ECP at our center were alternatively assigned, within the same ECP cycle (composed by two procedures each), to MNC collection with either device. Patients' characteristics, procedure, and product-related variables were compared. Results Thirty-nine patients (24 with GVHD and 15 with BOS) underwent a total of 126 ECP procedures, with good compliance to both devices. Product volume and platelet (PLT) and red blood cell contamination were significantly lower with the Spectra Optia. MNC collection efficiency (CE), purity, and PLT loss were similar between the two devices, while white blood cells CE was in favor of the COBE Spectra. Conclusion The Spectra Optia device proved to be a good option for MNC collection in the difficult ECP setting, since it ensures high-quality MNC collection, while at the same time saving personnel's time, guaranteeing optimal monitoring and care to this frail patient population. © 2013 American Association of Blood Banks.


PubMed | Humanitas Clinical and Research Center, National Diagnostics, University of Pavia, The International Center for Genetic Engineering and Biotechnology and Apheresis and Cell Therapy Unit
Type: Journal Article | Journal: Stem cells (Dayton, Ohio) | Year: 2016

Fibronectin (FN) is a major extracellular matrix protein implicated in cell adhesion and differentiation in the bone marrow (BM) environment. Alternative splicing of FN gene results in the generation of protein variants containing an additional EIIIA domain that sustains cell proliferation or differentiation during physiological or pathological tissue remodeling. To date its expression and role in adult hematopoiesis has not been explored. In our research, we demonstrate that during physiological hematopoiesis a small fraction of BM derived FN contains the EIIIA domain and that mice constitutively including (EIIIA(+/+) ) or excluding (EIIIA(-/-) ) the EIIIA exon present comparable levels of hematopoietic stem cells, myeloid and lymphoid progenitors within BM. Moreover, only minor alterations were detected in blood parameters and in hematopoietic frequencies of BM granulocytes/monocytes and B cells. As opposed to other tissues, unique compensatory mechanisms, such as increased FN accumulation and variable expression of the EIIIA receptors, Toll like receptor-4 and alpha9 integrin subunit, characterized the BM of these mice. Our data demonstrate that FN is a fundamental component of the hematopoietic tissue and that the EIIIA exon may play a key role in modulating hematopiesis in conditions of BM stress or diseases. Stem Cells 2016;34:2263-2268.

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