Jaakko Parkkinen

Non-transferrin-bound iron during allogeneic stem cell transplantation.

Hydroxyl radical formation catalysed by non‐transferrin‐bound iron (NTBI) might contribute to transplantation‐related complications. The occurrence of NTBI in 10 adult allogeneic stem cell transplantation (SCT) patients was followed for 20 d. The transferrin saturation reached 99% on d −4 and remained > 80% thereafter. NTBI, measured as bleomycin‐detectable iron, was detected for 6–18 d in all patients with a peak on d −4. High transferrin saturation levels were associated with the appearance of NTBI with a threshold at 80% saturation. Prevention of the potential deleterious effects of NTBI might reduce transplantation‐related morbidity.

A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance

Background and Objectives  The increasing demand for intravenous immunoglobulin (IVIG) necessitates the development of improved plasma fractionation methods, providing higher immunoglobulin G (IgG) recovery. Here, we describe a new IVIG production process resulting in a high yield of IgG and effective reduction of physico‐chemically resistant viruses.

Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients

Summary. Myeloablative treatment results in iron accumulation and the appearance of non‐transferrin‐bound iron (NTBI) in the circulation, which may contribute to treatment‐related organ damage and susceptibility to infections. The aim of this study was to investigate the efficacy of human apotransferrin in the binding of NTBI in patients receiving an allogeneic stem cell transplant after myeloablative conditioning. A single intravenous 100 mg/kg dose of apotransferrin was given to six adult patients on d 3 after the transplantation. Initially, all patients had serum transferrin saturation above 80% and NTBI in their serum. After the apotransferrin injection, serum NTBI became undetectable in all patients and transferrin saturation decreased to 30–50%. Serum transferrin increased by an average of 1·95 g/l. The administered apotransferrin was subsequently converted into monoferric and diferric transferrin forms. NTBI reappeared and transferrin saturation again exceeded 80% 12–48 h after the injection in four patients and after 6 d in one patient. NTBI remained non‐detectable for the whole 12 d follow‐up period in one patient. The apotransferrin injection was well tolerated and no adverse events with probable association with the apotransferrin were observed. Repeated apotransferrin infusions might completely eliminate NTBI and iron‐induced toxicity during myeloablative therapy.

Viral safety of Nanogam®, a new 15 nm‐filtered liquid immunoglobulin product

Background and Objectives  Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus‐reducing steps into the manufacturing process. Here we present virus‐elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam®) that comprises two specific virus‐reducing steps: a 15‐nm filtration step combined with pepsin treatment at pH 4·4 (pH 4·4/15NF); and solvent–detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus‐reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus‐reducing steps were studied.

Binding of Hepcidin to Plasma Proteins

To the Editor: The hepcidin hormone of 25 amino acid residues is a key regulator of iron homeostasis (1). Hepcidin has been shown to bind in vitro to α2-macroglobulin (α2M)1 and albumin in human plasma (2). It is not known, however, to what extent hepcidin is bound to proteins in vivo. For purposes of clinical interpretation, it is critical to know whether protein-bound or free hepcidin is being quantified. To characterize the binding of hepcidin to plasma proteins, we used gel filtration to fractionate 0.5 mL serum samples from both healthy individuals and patients undergoing hematopoietic stem cell transplantation. We similarly fractionated mixtures of hepcidin and α2M or albumin [1 g/L in 10 mmol/L potassium phosphate buffer, pH 7.4, containing 150 mmol/L NaCl (PBS)]. We used a Superdex™ 200 10/300 GL column (GE Healthcare Biosciences) equilibrated with PBS at a flow rate 0.5 mL/min. The column was calibrated with synthetic hepcidin (Peptide Institute), human albumin, and α2M. We collected 0.5-mL fractions and measured hepcidin in the fractions by HPLC–tandem mass spectrometry (LC-MS/MS) as previously described (3). We observed a single peak, corresponding to …

Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney tubular cells during cold storage.

Background. Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation. Methods. Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4°C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe. Results. Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG. Conclusion. The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.

Development of a Highly Purified Multicomponent Leukocyte IFN-α Product

A purification process was developed to obtain a human interferon- α (IFN-α) product that contains all major IFN-α subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-α in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 × 108 IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-α 1, IFN-α 2, IFN-α 8, IFN-α 10, IFN-α 14, IFN-α 17, and IFN-α 21. The subtypes IFN-α 4 and IFN-α 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilizat...

Inhibition of erythroid and granulocyte-macrophage colony formation by non-transferrin-bound iron in vitro: protective effect of apotransferrin.

Objectives:  The aim of the study was to investigate in vitro the effect of free iron on erythroid and granulocyte‐macrophage colony formation and the effect of binding free iron with apotransferrin.

Bicarbonate inhibits the growth of Staphylococcus epidermidis in platelet concentrates by lowering the level of non‐transferrin‐bound iron

BACKGROUND: Platelet concentrates (PCs) contain non‐transferrin‐bound iron (NTBI) owing to the displacement of iron from plasma‐derived transferrin by citrate. NTBI in the PC medium supports the growth of Staphylococcus epidermidis. The possibilities of lowering the level of NTBI have been studied with the aim to inhibit the growth of S. epidermidis in the PC medium.