Folke Knutson

A prospective, active haemovigilance study with combined cohort analysis of 19 175 transfusions of platelet components prepared with amotosalen–UVA photochemical treatment

A photochemical treatment process (PCT) utilizing amotosalen and UVA light (INTERCEPT™ Blood System) has been developed for inactivation of viruses, bacteria, parasites and leucocytes that can contaminate blood components intended for transfusion. The objective of this study was to further characterize the safety profile of INTERCEPT‐treated platelet components (PCT‐PLT) administered across a broad patient population.

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept? technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3–4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Improving the anti‐inflammatory effect of serum eye drops using allogeneic serum permissive for regulatory T cell induction

To investigate the cytokine composition and anti‐inflammatory effects of allogeneic serum preparations for improved use as serum eye drops.

Prophylactic platelet transfusions.

R. N. I. Pietersz, H. W. Reesink, S. Panzer, M. P. Gilbertson, M. E. Borosak, E. M. Wood, G. C. Leitner, W. Rabitsch, C. Ay, M. Lambermont, V. Deneys, D. Sondag, V. Compernolle, D. Legrand, A. François, R. Tardivel, F. Garban, R. B. Sawant, P. Rebulla, M. Handa, H. Ohto, J.-L. H. Kerkhoffs, A. Brand, E. Zhiburt, J. Cid, G. Escolar, M. Lozano, L. Puig, F. Knutson, H. Hallböök, N. Lubenow, L. Estcourt, S. Stanworth, M. F. Murphy, L. Williams, D.L. Mraz, R.L. Ross & E. Snyder

Photochemical pathogen inactivation of human serum enables its large-scale application in clinical cell transplantation

Stringent protocols for cGMP, issued by regulatorybodies in Asia, Europe and North America, regulateproduction of cells and tissues for clinical therapy. Dueto the risk of transferring infections, human serum, thepreferred supplement throughout the process of cell cul-ture and transplantation, cannot be used, resulting ininferior quality of released cell products. Photochemicaltreatment (INTERCEPT Cerus Europe BV, Amersfoort,Netherlands), with the psoralen compound, amotosalenHCl and long-wavelength ultraviolet light) has beenshown to inactivate any pathogen of RNA and DNAorigin [1–4]. INTERCEPT treatment of human serumcould allow its use in processing cells and tissues forclinical application.No long-term exposure in vitro of cells to residual amo-tosalen has been performed. However, a carcinogenicitystudy in p53 knockout mice exposed to doses 1000-foldthe clinical dose for 26 weeks, showed no evidence ofcarcogenicity [5].The use of PI-HS compared with C-HS was examined onhuman melanoma cell lines as well as in the production ofclinical grade islets of Langerhans (for the treatment ofType I Diabetes), mesenchymal stem cells (for the MSCs:treatment of GvHD grade III or IV) and T cells (for immunetherapy against malignant melanoma).There was no difference in rate of proliferation during a3-day culture in medium supplemented with either PI-HSor C-HS (10%) for five human melanoma cell-lines(397mel, 526mel, SK23mel, EB81mel, CP64mel).Similarly, there was no difference between islets culturedfor 4 days inCMRL-1066 supplemented with PI-HS orC-HSintermsofinsulinstimulationindexfromadynamicperifu-sion system where the average of the high values dividedwith the average of the low values (14AE7±4AE15, 10AE0±4AE57), the ADP⁄ATP-ratio (0AE010 ± 0AE013, )0AE0004 ±0AE013) and the capacity to cure STZ-diabetic mice (78%,87%). Likewise, no difference was found in intracellularinsulin content (2AE6±1AE14, 2AE3±0AE85 ng⁄ml), expressionof IL-6 (0AE0067 ± 0AE016, 0AE0075 ± 0AE018 pmol⁄lgDNA),IL-8 (0AE264 ± 0AE150, 0AE352 ± 0AE169 pmol⁄lgDNA), MCP-1(0AE164 ± 0AE086, 0AE166 ± 0AE080 pmol⁄lgDNA) or TissueFactor(0AE034 ± 0AE007,0AE087 ± 0AE057 pmol⁄lgDNA).Also, expansion of MSC and the capacity for immuno-suppression in MLC and PHA stimulated lymphocyte cul-tures (71 ± 13, 59 ± 9%) did not differ between MSCgenerated in CMRL-1066 supplemented with PI-HS orC-HS.Finally, no differences in regards of total number of Tcells generated (30AE9±3AE1, 29AE7±1AE5 millions⁄ml), foldexpansion (309AE4±31AE8, 297AE4±15AE2) or expression ofCD25, CD27 or CD26L were observed when T cells were cul-tured in RPMI-1640 supplemented with PI-HS or C-HS.Students t-test was used to evaluate the results between PIand control.The presented technique for PI of human serum providesa solution to an important safety and regulatory problem inmodern cell therapy. PI exerts no negative impact onhuman islets of Langerhans, MSCs, T cells or cell lines andcan even have a positive effect by down-regulation ofinflammatory mediators induced by DNA or RNA strandsreleased from damaged cells. INTERCEPT treatment ofhuman serum allows the routine use of human serum inclinical cell transplantation.In many European countries, the Intercept technology isroutinely used for PI of platelets and plasma for clinicaluse. Applying this already used technique in a new waycould be a new niche for blood banks. They can use this toprovide safer products for clinical cell culture and trans-plantation and by doing this increase the safety for thepatients.

Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units

BACKGROUNDnPlatelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units.nnnMATERIALS AND METHODSnPlatelet pooled units (nu2009=u200910) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ.nnnRESULTSnThe concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (pu2009<u20090.0001), 29.5% (pu2009=u20090.04) and 8.2% (pu2009=u20090.0004), respectively. A decrease was seen in the levels of IGF-1 and FGF-2 with 22% (pu2009=u20090.041) and 11% (pu2009=u20090.01), respectively. Cytokines were present only in very low concentrations and all other growth factors remained stable with time in storage.nnnCONCLUSIONnThe composition of mediators in platelet lysate obtained from pathogen-inactivated platelet units differs when produced from fresh and stored platelet units, respectively. This underscores the need for further standardization and optimization of this important product, which potentially may influence the clinical effects.

Inflammatory cytokines in serum samples of healthy blood donors

Blood components are essential in the arsenal of treatments inmodern health care and novel areas of use are being established, e.g. the use of platelet lysate to treat chronic ulcers [1] and serum for the treatment of dry eye [2]. It is conceivable that the efficacy of such novel blood products may vary considerably, since they are produced from a heterogeneous group of healthy donors. Particularly, standardizing and possibly optimizing the composition of complex products constitute a major undertaking, but may also improve their efficacy. Indeed, potential advantages of tailoring the composition of serumused as eye dropswere recently demonstrated to improve their anti-inflammatory properties [3]. At the same time, it was noted that the serum of healthy blood donors occasionally contains high levels of inflammatory cytokines. The latter may be undesired in a variety of clinical applications and possibly lead to adverse reactions [4]. We conducted a brief investigation aimed at assessing the frequency of an inflammatory cytokine profile in healthy blood donors, and analyzed 91 randomly selected regular donors at the Uppsala University Hospital Blood Bank, Sweden, for this purpose. Serumwas obtained as previously described [3] and analyzed for IFN-γ, TNF-α, CCL20, IL-1β, IL-6, IL-10, IL-12 p70, and IL-17A, using the Luminex xMAP technology (Merck Millipore, Darmstadt, Germany) according to themanufacturer’s instructions. The serawere somewhat arbitrarily stratified as containing low, intermediate or high levels of IFN-γ, defined as <100 ρg/mL, 100–300 ρg/mL and >300 ρg/mL, respectively. Of the 91 samples analyzed, 74 were categorized as low, 16 as intermediate and 1 as high (Fig. 1A). Linear regression demonstrated that all other cytokines correlated positively to the levels of IFN-γ (Fig. 1B). The serum classified as high was

Effect of Glyceryl Trinitrate on Staphylococcus aureus Growth and Leukocyte Activation during Simulated Extracorporeal Circulation

BACKGROUNDnPreviously, nitric oxide has been shown to possess antimicrobial effects. In this study, we aim to test the effect of glyceryl trinitrate (GTN) on Staphylococcus aureus growth during simulated extracorporeal circulation (SECC) and also to examine the effect of S. aureus, alone and in combination with GTN, on activation markers of the innate immune system during SECC.nnnMETHODSnIn an in vitro system of SECC, we measured GTN-induced changes in markers of leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth.nnnRESULTSnGTN had no effect on S. aureus growth after 240 minutes SECC. Staphylococcus aureus reduced the expression of granulocyte Fcγ-receptor CD32 but stimulated the expression of monocyte CD32. Staphylococcus aureus stimulated expression of some leukocyte adhesion key proteins, activation marker CD66b, lipopolysaccharide-receptor CD14, and C3b-receptor CD35. Staphylococcus aureus and GTN addition induced significant increases in monocyte CD63 (lysosomal granule protein) levels.nnnCONCLUSIONnGTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation.

Enhanced Growth of Staphylococcus aureus after Nitric Oxide Supplementation during Simulated Extracorporeal Circulation

BACKGROUNDnSeveral factors contribute to postoperative bacterial infections in cardiac surgery. Long operation times and the use of extracorporeal circulation increase the risk of infection. Nitric oxide has been shown to possess a broad spectrum antimicrobial effect.nnnMETHODSnIn this study, we investigated the effect of nitric oxide on S. AUREUS growth in whole blood during simulated extracorporeal circulation.nnnRESULTSnS. AUREUS growth increased 6.2-fold after 180 min SECC in the presence of nitric oxide. Leukocyte counts remained unchanged without any differences between the groups. We observed a steady increase in markers of oxidative stress and activity of the innate immune system. Myeloperoxidase levels increased 8-fold, and C3a and terminal complement complex by 2-fold after 180 min.nnnCONCLUSIONnS. AUREUS growth is not due to the effect of nitric oxide on the innate immune system but from its effect on the bacteria itself. It has been shown that nitric oxide stimulates the expression of inducible lactate dehydrogenase, specific to S. AUREUS, which improves its resistance to oxidative stress, and may give S. AUREUS a survival advantage resulting in increased growth.